Microscope-based systems and methods using ultraviolet light permeable lenses

Abstract

The invention provides a microscope-based system and method for image-guided microscopic illumination. The microscope includes a stage, and the stage is configured to load a sample. A microscope-based system includes a first light source, a second light source, a first dichroic mirror, a second dichroic mirror, and a receiver. The first dichroic mirror and the second dichroic mirror have high penetrability in a transmission wave band and high reflectivity in a reflection wave band so as to selectively transmit / receive a light beam from the first light source to a sample and a light beam from the first light source to the receiver for sample imaging; and allowing the light beam to be transmitted from the second light source to the sample for photochemical processing (image irradiation) at a specific location according to the imaging. The characteristic design of these two dichroic mirror systems allows multi-frequency (wavelength) imaging and image illumination to be operated quickly without employing mechanical switches to switch the different optical paths for each individual function.

Description

Priority claim

[0001] The present invention claims priority to U.S. Provisional Patent Application No. 63 / 354,806, filed on June 23, 2022, entitled “Microscope-Based Systems and Methods Using Ultraviolet Penetrable Lenses,” and U.S. Provisional Patent Application No. 63 / 509,485, filed on June 21, 2023, entitled “Microscope-Based Systems and Methods Using Ultraviolet Penetrable Lenses,” and the entire contents of these two U.S. Provisional Applications are incorporated herein by reference. Incorporated herein by reference

[0002] All publications and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Background Art

[0003] The present invention relates to a system and method for illuminating an image on a sample, and more particularly to a microscope system and method for illuminating different images continuously at high speed in a large number of fields of view.

[0004] There is a need to image specific locations in samples (e.g., biological samples). For example, photobleaching of molecules in certain subcellular regions, photoactivation of fluorophores in defined locations, optogenetics, phototriggered release of reactive oxygen species in specified organelles, or photoinduced labeling of biomolecules in defined structural features of cells require image illumination. For some applications, the images in the above processing may need to be determined by microscopic images. Some applications require processing of sufficient samples, adding high-content requirements to repeat the processing in multiple areas. Systems that can perform such automated image-based local phototriggered processing are rare.

[0005] One example of processing proteins, lipids or nucleic acids is to label them for separation and identification. Labeled proteins, lipids or nucleic acids can be separated and identified using other systems such as mass spectrometers or sequencers. The STOMP (Space Target Optical Microproteomics) technology proposed by Kevin CHadley et al. in 2015 is a manually operated technology using a commercially available two-photon system, but lacks the main elements of the present invention to achieve high-throughput capabilities. Laser capture microdissection (LCM) systems that use laser cutting and are widely used to separate parts of tissues or cell cultures do not have the axial precision that can be achieved by the present invention in addition to lacking high-throughput capabilities. Summary of the invention

[0006] A microscope-based illumination and imaging system comprises: a first light source; a first dichroic mirror, which is suitable for reflecting a light beam from the first light source onto a sample; and a second light source, which is suitable for transmitting a light beam with one or more wavelengths falling within a wavelength range of the second light source through a tube lens of the microscope, the second dichroic mirror, and the first dichroic mirror to the sample, wherein the wavelength range of the second light source has a lower limit value equal to or greater than 250 nm and an upper limit value equal to or less than 470 nm, and the second dichroic mirror is suitable for reflecting the light beam from the sample.

[0007] In one aspect, the second dichroic mirror has high transmittance in at least a second dichroic mirror transmission band over a second light source wavelength range.

[0008] In one aspect, the second dichroic mirror transmission band extends above and / or below the wavelength range of the second light source.

[0009] In one aspect, the second dichroic mirror has high transmittance in a second dichroic mirror transmission band having a lower limit value equal to or greater than 250 nm and an upper limit value equal to or less than 470 nm.

[0010] In one aspect, the second dichroic mirror has a high reflectivity in a second dichroic mirror reflection band that does not overlap with the second dichroic mirror transmission band.

[0011] In one aspect, the second dichroic mirror reflection wavelength band has a lower limit value equal to or greater than 350 nm and an upper limit value equal to or less than 900 nm.

[0012] In one aspect, the first dichroic mirror has high transmittance in a first dichroic mirror transmission band of at least the second light source wavelength range.

[0013] In one aspect, the first dichroic mirror transmission band extends above and / or below the wavelength range of the second light source.

[0014] In one aspect, the first dichroic mirror has high transmittance in a primary first dichroic mirror transmission band having a lower limit value equal to or greater than 250 nm and an upper limit value equal to or less than 470 nm.

[0015] In one aspect, the first dichroic mirror has high transmittance in a secondary first dichroic mirror transmission band, the secondary first dichroic mirror transmission band (i) having a lower limit value equal to or greater than 350 nm and having an upper limit value equal to or less than 470 nm, and (ii) not overlapping with the primary first dichroic mirror transmission band.

[0016] In one aspect, the first dichroic mirror has high transmittance in a secondary first dichroic mirror transmission band, the secondary first dichroic mirror transmission band (i) having a lower limit value equal to or greater than 440 nm and having an upper limit value equal to or less than 570 nm and (ii) not overlapping with the primary first dichroic mirror transmission band.

[0017] In one aspect, the first dichroic mirror has high transmittance in a secondary first dichroic mirror transmission band, the secondary first dichroic mirror transmission band (i) having a lower limit value equal to or greater than 500 nm and having an upper limit value equal to or less than 650 nm.

[0018] In one aspect, the first dichroic mirror has high transmittance in a secondary first dichroic mirror transmission band, the secondary first dichroic mirror transmission band (i) having a lower limit value equal to or greater than 600 nm and having an upper limit value equal to or less than 750 nm.

[0019] In one aspect, the first dichroic mirror has high transmittance in a secondary first dichroic mirror transmission band, the secondary first dichroic mirror transmission band (i) having a lower limit value equal to or greater than 700 nm and having an upper limit value equal to or less than 900 nm.

[0020] In one aspect, the first dichroic mirror has high transmittance in a plurality of different and non-overlapping transmission bands, each of the transmission bands having a lower limit equal to or greater than 350 nm and having an upper limit equal to or less than 900 nm.

[0021] In one aspect, the first dichroic mirror has high reflectivity in a first dichroic mirror reflection band, which (i) has a lower limit value equal to or greater than 300 nm and an upper limit value equal to or less than 420 nm, and (ii) does not overlap with the primary first dichroic mirror transmission band.

[0022] In one aspect, the first dichroic mirror has high reflectivity in a first dichroic mirror reflection band, which (i) has a lower limit value equal to or greater than 430nm and an upper limit value equal to or less than 530nm, and (ii) does not overlap with the primary first dichroic mirror transmission band.

[0023] In one aspect, the first dichroic mirror has high reflectivity in a first dichroic mirror reflection band having a lower limit value equal to or greater than 480 nm and having an upper limit value equal to or less than 570 nm.

[0024] In one aspect, the first dichroic mirror has high reflectivity in a first dichroic mirror reflection band having a lower limit value equal to or greater than 530 nm and having an upper limit value equal to or less than 610 nm.

[0025] In one aspect, the first dichroic mirror has high reflectivity in a first dichroic mirror reflection band having a lower limit value equal to or greater than 610 nm and an upper limit value equal to or less than 670 nm.

[0026] In one aspect, the first dichroic mirror has high reflectivity in a first dichroic mirror reflection band having a lower limit value equal to or greater than 710 nm and having an upper limit value equal to or less than 770 nm.

[0027] In one aspect, the first dichroic mirror has high reflectivity in a plurality of different and non-overlapping first dichroic mirror reflection bands, each of which (i) has a lower limit value equal to or greater than 350 nm and an upper limit value equal to or less than 900 nm, and (ii) does not overlap with the primary first dichroic mirror transmission band.

[0028] In one aspect, the sample is disposed on a stage of the microscope.

[0029] In one aspect, the system further comprises a receiver adapted to receive the light beam reflected by the second dichroic mirror.

[0030] In one aspect, the receiver is a camera.

[0031] Provided is an illumination and imaging system based on a microscope, comprising: a first light source; a first dichroic mirror, adapted to reflect a light beam from the first light source; a second dichroic mirror, adapted to transmit the light beam from the first light source to a sample; and a second light source, adapted to transmit one or more light beams with wavelengths falling within a wavelength range of the second light source through a tube lens of the microscope, the first dichroic mirror and the second dichroic mirror to the sample, wherein the wavelength range of the second light source has a lower limit value equal to or greater than 250 nm and an upper limit value equal to or less than 470 nm, and the second dichroic mirror is adapted to reflect the light beam from the sample.

[0032] In one aspect, the first dichroic mirror has high transmittance in a first dichroic mirror transmission band of at least the second light source wavelength range.

[0033] In one aspect, the first dichroic mirror transmission band extends above and / or below the wavelength range of the second light source.

[0034] In one aspect, the first dichroic mirror has high transmittance in a first dichroic mirror transmission band, the first dichroic mirror transmission band having a lower limit value equal to or greater than 250 nm and an upper limit value equal to or less than 470 nm.

[0035] In one aspect, the first dichroic mirror has high reflectivity in a first dichroic mirror reflection band that does not overlap with the first dichroic mirror transmission band.

[0036] In one aspect, the first dichroic mirror reflection wavelength band has a lower limit value equal to or greater than 300 nm and an upper limit value equal to or less than 900 nm.

[0037] In one aspect, the second dichroic mirror has high transmittance in a second dichroic mirror transmission band of at least the second light source wavelength range.

[0038] In one aspect, the second dichroic mirror transmission band extends above and / or below the wavelength range of the second light source.

[0039] In one aspect, the second dichroic mirror has high transmittance in a primary second dichroic mirror transmission band having a lower limit value equal to or greater than 250 nm and having an upper limit value equal to or less than 530 nm.

[0040] In one aspect, the second dichroic mirror has high transmittance in a secondary second dichroic mirror transmission band, the secondary second dichroic mirror transmission band (i) having a lower limit value equal to or greater than 430 nm and having an upper limit value equal to or less than 530 nm and (ii) not overlapping with the primary first dichroic mirror transmission band.

[0041] In one aspect, the second dichroic mirror has high transmittance in a secondary second dichroic mirror transmission band having a lower limit value equal to or greater than 480 nm and an upper limit value equal to or less than 570 nm.

[0042] In one aspect, the second dichroic mirror has high transmittance in a secondary second dichroic mirror transmission band having a lower limit value equal to or greater than 530 nm and having an upper limit value equal to or less than 610 nm.

[0043] In one aspect, the second dichroic mirror has high transmittance in a secondary second dichroic mirror transmission band having a lower limit value equal to or greater than 610 nm and having an upper limit value equal to or less than 670 nm.

[0044] In one aspect, the second dichroic mirror has high transmittance in a secondary second dichroic mirror transmission band having a lower limit value equal to or greater than 710 nm and having an upper limit value equal to or less than 770 nm.

[0045] In one aspect, the second dichroic mirror has high transmittance in a plurality of different and non-overlapping transmission bands, each of the transmission bands having a lower limit equal to or greater than 400 nm and an upper limit equal to or less than 900 nm.

[0046] In one aspect, the second dichroic mirror has high reflectivity in a second dichroic mirror reflection band, which (i) has a lower limit value equal to or greater than 350nm and an upper limit value equal to or less than 470nm and (ii) does not overlap with the primary second dichroic mirror transmission band.

[0047] In one aspect, the second dichroic mirror has high reflectivity in a second dichroic mirror reflection band, which (i) has a lower limit value equal to or greater than 440nm and an upper limit value equal to or less than 570nm and (ii) does not overlap with the primary second dichroic mirror transmission band.

[0048] In one aspect, the second dichroic mirror has high reflectivity in a second dichroic mirror reflection band having a lower limit value equal to or greater than 500 nm and having an upper limit value equal to or less than 650 nm.

[0049] In one aspect, the second dichroic mirror has high reflectivity in a second dichroic mirror reflection band having a lower limit value equal to or greater than 600 nm and having an upper limit value equal to or less than 750 nm.

[0050] In one aspect, the second dichroic mirror has high reflectivity in a second dichroic mirror reflection band having a lower limit value equal to or greater than 700 nm and having an upper limit value equal to or less than 900 nm.

[0051] In one aspect, the second dichroic mirror has high reflectivity in a plurality of different and non-overlapping second dichroic mirror reflection bands, each of which (i) has a lower limit value equal to or greater than 300 nm and an upper limit value equal to or less than 900 nm, and (ii) does not overlap with the primary second dichroic mirror transmission band.

[0052] In one aspect, the sample is disposed on a stage of the microscope.

[0053] In one aspect, the system includes a receiver adapted to receive the light beam reflected by the second dichroic mirror.

[0054] In one aspect, the receiver is a camera.

[0055] A method is provided, comprising: projecting a light beam of a first light source wavelength from a first light source to a first dichroic mirror to reflect the light beam onto a sample; causing a light beam reflected by or generated by the sample in response to the first light source to travel through the first dichroic mirror to a second dichroic mirror to reflect the light beam to a receiver; and projecting a light beam of a second light source wavelength range from a second light source through the second dichroic mirror and through the first dichroic mirror onto the sample.

[0056] In one aspect, the first light source wavelength comprises a first light source wavelength range.

[0057] In one aspect, the first light source wavelength comprises approximately 488 nm.

[0058] In one aspect, the first light source wavelength comprises approximately 561 nm.

[0059] In one aspect, the first light source wavelength comprises approximately 635 nm.

[0060] In one aspect, the second light source has a wavelength range comprising approximately 250 to 470 nm.

[0061] In one aspect, the second light source has a wavelength range suitable for excitation, photoactivation, photo-manipulation or other photochemical processing of the sample.

[0062] In one aspect, the first dichroic mirror and the second dichroic mirror have high transmittance in the second light source wavelength range of the second light source.

[0063] In one aspect, the first dichroic mirror and the second dichroic mirror transmit more than 80% of the light beam from the second light source.

[0064] In one aspect, the second dichroic mirror has a high reflectivity in a second dichroic mirror reflection band that does not overlap with the second dichroic mirror transmission band.

[0065] In one aspect, the second dichroic mirror reflection wavelength band has a lower limit value equal to or greater than 350 nm and an upper limit value equal to or less than 900 nm.

[0066] In one aspect, the first dichroic mirror has high transmittance in a first dichroic mirror transmission band of at least the second light source wavelength range.

[0067] In one aspect, the receiver is a camera.

[0068] A method is provided, comprising: projecting a light beam of a first light source wavelength from a first light source to a first dichroic mirror to reflect the light beam through a second dichroic mirror onto a sample; passing a light beam reflected by or generated by the sample in response to the first light source to the second dichroic mirror to reflect the light beam to a receiver; and projecting a light beam of a second light source wavelength range from a second light source through the first dichroic mirror and through the second dichroic mirror onto the sample.

[0069] In one aspect, the first light source wavelength comprises a first light source wavelength range.

[0070] In one aspect, the first light source wavelength comprises approximately 488 nm.

[0071] In one aspect, the first light source wavelength comprises approximately 561 nm.

[0072] In one aspect, the first light source wavelength comprises approximately 635 nm.

[0073] In one aspect, the second light source has a wavelength range comprising approximately 250 to 470 nm.

[0074] In one aspect, the second light source has a wavelength range suitable for excitation, photoactivation, photomanipulation, or other photochemical processing of the sample.

[0075] In one aspect, the first dichroic mirror and the second dichroic mirror have high transmittance in the second light source wavelength range of the second light source.

[0076] In one aspect, the first dichroic mirror and the second dichroic mirror transmit more than 80% of the light beam from the second light source.

[0077] In one aspect, the receiver is a camera.

[0078] In one aspect, the systems and methods described herein do not include a mechanical switch for switching the path of light from the first light source or the second light source. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] The embodiments can be more fully understood with reference to the specific implementation manner and the accompanying drawings, which are only for illustration and not for limiting the present invention, and in the accompanying drawings:

[0080] Figure 1 A microscope system is shown for illuminating a sample on a stage for imaging and / or sample photoactivation.

[0081] Figure 2 is a graph showing the transmission and reflection characteristics of the first dichroic mirror.

[0082] Figure 3 is a graph showing the transmission and reflection characteristics of the second dichroic mirror.

[0083] Figure 4 Another embodiment of a microscope system for illuminating a sample on a stage for imaging and / or sample photoactivation is shown.

[0084] Figure 5 is a graph showing the transmission and reflection characteristics of the second dichroic mirror.

[0085] Figure 6 is a graph showing the transmission and reflection characteristics of the first dichroic mirror. DETAILED DESCRIPTION

[0086] A microscope-based system and method for image-guided microscopic illumination are provided. The microscope system may include a stage configured to load a sample. The microscope system may include a first light source, a second light source, a first dichroic mirror, a second dichroic mirror, and a receiver. The first and second dichroic mirrors may have high transmittance in a transmission band and high reflectivity in a reflection band to selectively transmit / receive a light beam from the first light source to the sample and a light beam to the receiver for sample imaging, and to allow a light beam from the second light source to be transmitted to the sample for photochemical treatment (image illumination) at a specific location based on imaging. The characteristic design of the two dichroic mirror systems allows multi-frequency (wavelength) imaging and image illumination, which can be quickly operated without using a mechanical switch to switch different light paths for each individual function, to achieve efficient image-guided microscopic illumination for phototreatment of a large number of biomolecules in the sample, which can improve the sensitivity of the results and be used for further analysis.

[0087] Figure 1A microscope system 10 is shown for illuminating a sample 12 on a stage 14 for imaging and / or sample photoactivation. A first light source 16 projects a beam of light (e.g., having a wavelength of 488 nm, 561 nm, or 635 nm) to a first dichroic mirror 18, which reflects the beam through an objective 20 of the microscope onto the sample 12. The signal from the sample (i.e., the beam of light reflected by or generated by the sample in response to the beam from the first light source 16) then passes through the objective 20 and through the first dichroic mirror 18 to a second dichroic mirror 22, which reflects the signal to an eyepiece, camera, or other receiver 24. The beam from the first light source 16 can be used to obtain an image of the sample and / or for photoactivation of the sample.

[0088] The system 10 also has a second light source 26 (e.g., one or more wavelengths in the range of 250 to 470 nm) for use, for example, in excitation, photoactivation, photo-manipulation, or other photochemical treatment of the sample 12. The light source 26 transmits a beam of light through the microscope's tube lens 28, the second dichroic mirror 22, the first dichroic mirror 18, and the objective lens 20 to the sample 12. The sample signal generated by illumination by the light source 26 again passes through the objective lens 20 and through the first dichroic mirror 18 to the second dichroic mirror 22, which reflects the signal to the eyepiece, camera, or other receiver 24.

[0089] Figure 2 is a graph showing the transmission and reflection characteristics of the first dichroic mirror 18, and Figure 3Graphs showing the transmission and reflection characteristics of the second dichroic mirror 22. In order to transmit the light beam from the light source 26, the first dichroic mirror 18 and the second dichroic mirror 22 have high transmission (i.e., transmit more than 80% or more than 90% of the incident light) in a wavelength range at least equal to, and possibly extending above and / or below, the wavelength range of the second light source 26. The first dichroic mirror 18 also has high transmittance at wavelengths corresponding to the expected images and signals from the sample (e.g., wavelengths of about 502.5-544.5 nm for fluorescent dyes Alexa 488 or ATTO 488 or green fluorescent protein (GFP) or fluorescein isothiocyanate (FITC) or yellow fluorescent protein (YFP) in the imaged sample, wavelengths of 582-617.5 nm for fluorescent dyes Alexa 568 or Alexa 594 or ATTO 550 or ATTO 565 or ATTO 590 or Cy3 or Cy3B or tetramethylrhodamine isothiocyanate (TRITC) or red fluorescent protein (RFP) or mCherry (red fluorescent dye) or Texas Red (red fluorescent dye) in the imaged sample, and wavelengths of about 582-617.5 nm for fluorescent dyes Alexa 568 or Alexa 594 or ATTO 550 or ATTO 565 or ATTO 590 or Cy3 or Cy3B or tetramethylrhodamine isothiocyanate (TRITC) or red fluorescent protein (RFP) or mCherry (red fluorescent dye) or Texas Red (red fluorescent dye) in the imaged sample). 647 or Cy5 wavelength 663-700nm), so that the image and / or signal from the sample can travel through the first dichroic mirror 18 to the second dichroic mirror 22, and in the wavelength range covering the wavelength of the first light source 16 (for example: the wavelength of about 473-491nm for the fluorescent dye Alexa 488 or ATTO 488 or green fluorescent protein (GFP) or fluorescein isothiocyanate (FITC) or yellow fluorescent protein (YFP) used in the imaging sample, the fluorescent dye Alexa568 or Alexa594 or ATTO 550 or ATTO 565 or ATTO 590, Cy3, Cy3B or tetramethylrhodamine isothiocyanate (TRITC) or red fluorescent protein (RFP) or mCherry (red fluorescent dye) or Texas The first dichroic mirror 18 has a high reflectivity (i.e., reflects more than 80% or more than 90% of the light beam) at wavelengths of about 559-568.2 nm for Red (red fluorescent dyes), and 632-647.1 nm for fluorescent dyes Alexa647 or Cy5 in the imaging sample. The second dichroic mirror 22 has a high reflectivity at wavelengths of about 470-900 nm, so that the signal from the sample (e.g., from imaging (fluorescence imaging, bright field imaging, dark field imaging, differential interference contrast (DIC) imaging, phase contrast imaging) or from photoactivation (fluorescence, reflection, bleaching, spectral signals)) is directed to the eyepiece, camera or other receiver 24.

[0090] In some embodiments, details of the first dichroic mirror 18 and the second dichroic mirror 22 can be listed in Table 1.

[0091] Table 1

[0092] Figure 4 Another embodiment of a microscope system 40 for illuminating a sample 12 on a stage 14 for imaging and / or sample photoactivation is shown. A first light source 42 projects a beam of light (e.g., at a wavelength of 488 nm, 561 nm, or 635 nm) to a first dichroic mirror 44, which reflects and directs the beam through a second dichroic mirror 46 and the objective lens 20 of the microscope onto the sample 12. The signal from the sample (i.e., the beam of light reflected by or generated by the sample in response to the beam from the light source 42) then passes through the objective lens 20 and is reflected by the second dichroic mirror 46 to an eyepiece, camera, or other receiver 48. The beam from the first light source 42 can be used to obtain an image of the sample and / or for sample photoactivation.

[0093] The system 40 also has a second light source 50 (e.g., one or more wavelengths in the range of 250 to 470 nm) for use, for example, in excitation, photoactivation, photomanipulation, or other photochemical treatment of the sample 12. The light source 50 transmits a beam of light through the microscope's tube lens 28, the first dichroic mirror 44, the second dichroic mirror 46, and the objective 20 to the sample 12. The sample signal resulting from illumination by the light source 50 travels again through the objective 20 and is reflected by the second dichroic mirror 46 to the eyepiece, camera, or other receiver 48.

[0094] Figure 5 is a graph showing the transmission and reflection characteristics of the second dichroic mirror 46, and Figure 6Graphs showing the transmission and reflection characteristics of the first dichroic mirror 44. In order to transmit the light beam from the light source 50, the first dichroic mirror 44 and the second dichroic mirror 46 have high transmission (i.e., transmit more than 80% or more than 90% of the incident light) in a wavelength range at least equal to, and possibly extending above and / or below, the wavelength range of the second light source 50. The second dichroic mirror 46 also has high transmittance in a wavelength range covering multiple wavelengths of the first light source 42 (for example: a wavelength of approximately 473-491 nm for fluorescent dyes Alexa 488 or ATTO 488 or GFP or FITC or YFP used in imaging samples, a wavelength of 559-568.2 nm for fluorescent dyes Alexa 568 or Alexa 594 or ATTO 550 or ATTO 565 or ATTO 590, Cy3, or Cy3B or TRITC or RFP or mCherry or Texas Red used in imaging samples, and a wavelength of 632-647.1 nm for fluorescent dyes Alexa 647 or Cy5 used in imaging samples), while the first dichroic mirror has high reflectivity in these wavelength ranges. The second dichroic mirror 46 is configured to image the sample at wavelengths corresponding to the desired image and signal from the sample (e.g., wavelengths of about 502.5-544.5 nm for the fluorescent dyes Alexa 488 or ATTO 488 or GFP or FITC or YFP in the imaged sample, wavelengths of 582-617.5 nm for the fluorescent dyes Alexa 568 or Alexa 594 or ATTO 550 or ATTO 565 or ATTO 590, Cy3 or Cy3B, or TRITC, or RFP, or mCherry, or Texas Red in the imaged sample, and wavelengths of about 582-617.5 nm for the fluorescent dyes Alexa 568 or Alexa 594 or ATTO 550 or ATTO 565 or ATTO 590, Cy3 or Cy3B, or TRITC, or RFP, or mCherry, or Texas Red in the imaged sample). 647 or Cy5 (wavelength 663-700nm) is highly reflective, so that images and / or signals from the sample (for example, from imaging (fluorescence imaging, bright field imaging, dark field imaging, differential interference contrast (DIC) imaging, phase contrast imaging) or from photoactivation (fluorescence, reflection, fading, spectral signals)) are directed to the eyepiece, camera or other receiver 48.

[0095] In some embodiments, Figure 5 In the embodiment, the primary second dichroic mirror transmission band is 350nm to 491nm, which is higher than the wavelength range of the second light source (250-470nm). In this embodiment, the primary second dichroic mirror transmission band can be used to (1) transmit the second light source (e.g., 360nm or 405nm) and (2) transmit the first light source (e.g., 473nm-491nm) for exciting the fluorescent dyes Alexa 488 or Atto 488 or GFP or FITC in the sample, and its emission light will fall within the range of the reflection band of the second dichroic mirror (502.5-544.5nm).

[0096] In some embodiments, the details of the first dichroic mirror 44 and the second dichroic mirror 46 can be listed in Table 2.

[0097] Table 2

[0098] In some embodiments, dichroic mirrors 22 and 46 are 24-26 mm by 34-37 mm by 1-2 mm and have a normal radius of curvature greater than or equal to 100 meters and a reflected wavefront error less than 2λP-VRWE.

[0099] Here, when a feature or element is referred to as being "on" another feature or element, it can be directly on the other features or elements or there can be other intermediate features and / or elements. Conversely, when a feature or element is referred to as being "directly" located "on" another feature or element, there are no intermediate features or elements. It should also be understood that when a feature or element is referred to as being "connected," "attached" or "coupled" to another feature or element, it can be directly "connected," "attached" or "coupled" to the other features or elements or there can be intermediate features or elements. Conversely, when a feature or element is referred to as being "directly connected," "directly attached" or "directly coupled" to another feature or element, there are no intermediate features or elements. Although the features and elements described or shown are with respect to one embodiment, the features and elements described or shown may be applied to other embodiments. It is also understood by those skilled in the art that a structure or feature referred to as being "adjacent" to another feature may have a portion that overlaps with or is located below the adjacent feature.

[0100] The technical terms used herein are only used for the purpose of describing specific embodiments and are not intended to limit the present invention. For example, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" used herein are also intended to include multiple situations. It should also be understood that when the terms "include" and / or "include" are used in this specification, they indicate the presence of the stated features, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components and / or combinations thereof. The term "and / or" as used herein includes any and all combinations of one or more related enumerated items and can be abbreviated as " / ".

[0101] In order to facilitate the description of the relative relationship between one element or feature and another element or feature as shown in the figure, spatially related terms may be used herein, such as: "below", "low", "lower", "above", "upper", etc. It should be understood that in addition to the orientation depicted in the figure, these spatially related terms are also intended to cover different orientations of the device in use or operation. For example, if the device in the figure is turned over, the elements described as "below" or "below" other elements or features will be directed to "above" other elements or features. Therefore, the exemplary term "below" can cover both upper and lower orientations. The device can be directed to other orientations (rotated 90 degrees or in other orientations) and the spatially related descriptions used herein are interpreted accordingly. Similarly, unless specifically indicated, the terms "upwardly", "downwardly", "vertical", "horizontally", etc. in this article are for illustrative purposes only.

[0102] Although the terms "first" and "second" are used herein to describe different features / elements (including steps), these features / elements should not be limited by these terms unless the context indicates otherwise. These terms can be used to distinguish one feature / element from another feature / element. Therefore, the first feature / element discussed below can be referred to as the second feature / element, and similarly, the second feature / element discussed below can be referred to as the first feature / element without departing from the teachings of the present invention.

[0103] In this specification and the appended claims, unless the context requires otherwise, the word "includes", and variations such as "comprises" and "comprisng", refer to various components that can be used together in methods and articles (e.g., compositions and devices including elements and methods). For example, the term "comprising" will be understood to imply the inclusion of any stated elements or steps, but not the exclusion of any other elements or steps.

[0104] Unless otherwise specifically specified, all numerical values ​​used in the specification and patent application range herein, including in the examples, can be interpreted as beginning with the word "about" or "approximately", even if the term does not explicitly appear. The term "about" or "approximately" can also be used when describing the size and / or position to indicate that the value and / or position described is within the reasonable expected range of the value and / or position. For example, the numerical value can be a value of ±0.1% of the value (or range of values), a value of ±1% of the value (or range of values), a value of ±2% of the value (or range of values), a value of ±5% of the value (or range of values), a value of ±10% of the value (or range of values), etc. Unless otherwise specified in the context, any numerical value given herein should also be understood to have approximately or approximately the numerical value. For example, if the disclosed value is "10", the value of "approximately 10" is also disclosed. Any numerical range cited herein is intended to include all sub-ranges contained in the numerical range. It should also be understood that when a value is disclosed, as will be understood by those skilled in the art, "less than or equal to" the value, "greater than or equal to the value," and possible ranges between values ​​are also disclosed. For example, if the value "X" is disclosed, then "less than or equal to "X"" and "greater than or equal to "X"" (e.g., when "X" is a numerical value) are also disclosed. It should also be understood that in this application, data is provided in a variety of different formats, and that the data represents endpoints and starting points and ranges of any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it should be understood that greater than, greater than or equal to, less than, less than or equal to, and equal to 10 and 15, as well as between 10 and 15, are all considered disclosed. It should also be understood that every unit between two specific units is also disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

[0105] Although various different embodiments are described above, any of a variety of changes may be made to the different embodiments without departing from the scope of the invention as described in the claims. For example, in alternative embodiments, the order of execution of various described method steps can often be changed, and in other alternative embodiments, one or more method steps can be skipped altogether. Various components and optional features of system embodiments may be included in some embodiments but not in other embodiments. Therefore, the previous description is primarily for exemplary purposes and should not be construed as limiting the scope of the invention set forth in the claims.

[0106] The examples and descriptions contained herein show specific embodiments in which the subject matter may be implemented in an illustrative and non-limiting manner. As described above, other embodiments may be utilized and derived therefrom, so that structural and logical replacements and changes may be made without departing from the scope of the present disclosure. If in fact multiple inventions or inventive concepts are disclosed, such embodiments of the subject matter of the present invention may be referred to herein individually or collectively by the term "invention", which is only for convenience and is not intended to limit the scope of the present application to any single invention or inventive concept. Therefore, although specific embodiments have been illustrated and described herein, any configuration calculated to achieve the same purpose may replace the specific embodiments shown. The present disclosure is intended to cover any and all modifications or variations of the various embodiments. After reading the above description, it will be clear to those skilled in the art that the above embodiments and other embodiments not specifically described herein will be combined.

Claims

1. A microscope-based illumination and imaging system, comprising: The first light source; a first dichroic mirror, adapted to reflect the light beam from the first light source onto a sample; as well as a second light source, adapted to transmit one or more light beams having wavelengths falling within a second light source wavelength range through a tube lens of a microscope, a second dichroic mirror, and the first dichroic mirror to the sample, wherein the second light source wavelength range has a lower limit value equal to or greater than 250 nm and an upper limit value equal to or less than 470 nm, and the second dichroic mirror is adapted to reflect the light beam from the sample.

2. The system according to claim 1, wherein: The second dichroic mirror has high transmittance in a second dichroic mirror transmission band of at least the second light source wavelength range.

3. The system according to claim 2, wherein: The transmission wavelength band of the second dichroic mirror extends above and / or below the wavelength range of the second light source.

4. The system according to claim 1, wherein: The second dichroic mirror has high transmittance in a second dichroic mirror transmission band, and the second dichroic mirror transmission band has a lower limit value equal to or greater than 250 nm and an upper limit value equal to or less than 470 nm.

5. The system according to claim 4, wherein: The second dichroic mirror has high reflectivity in a second dichroic mirror reflection band, and the second dichroic mirror reflection band does not overlap with the second dichroic mirror transmission band.

6. The system according to claim 5, wherein: The second dichroic mirror reflection wavelength band has a lower limit value equal to or greater than 350 nm and an upper limit value equal to or less than 900 nm.

7. The system according to claim 1, wherein: The first dichroic mirror has high transmittance in a first dichroic mirror transmission band of at least the second light source wavelength range.

8. The system according to claim 7, wherein: The transmission wavelength band of the first dichroic mirror extends above and / or below the wavelength range of the second light source.

9. The system according to claim 1, wherein: The first dichroic mirror has high transmittance in a primary first dichroic mirror transmission band, the primary first dichroic mirror transmission band having a lower limit value equal to or greater than 250 nm and having an upper limit value equal to or less than 470 nm.

10. The system according to claim 9, wherein: The first dichroic mirror has high transmittance in a secondary first dichroic mirror transmission band, the secondary first dichroic mirror transmission band (i) has a lower limit value equal to or greater than 350 nm and an upper limit value equal to or less than 470 nm, and (ii) does not overlap with the primary first dichroic mirror transmission band.

11. The system according to claim 9, wherein: The first dichroic mirror has high transmittance in a secondary first dichroic mirror transmission band, the secondary first dichroic mirror transmission band (i) has a lower limit value equal to or greater than 440 nm and has an upper limit value equal to or less than 570 nm and (ii) does not overlap with the primary first dichroic mirror transmission band.

12. The system according to claim 9, wherein: The first dichroic mirror has high transmittance in a secondary first dichroic mirror transmission band, and the secondary first dichroic mirror transmission band (i) has a lower limit value equal to or greater than 500 nm and an upper limit value equal to or less than 650 nm.

13. The system according to claim 9, wherein: The first dichroic mirror has high transmittance in a secondary first dichroic mirror transmission band, and the secondary first dichroic mirror transmission band (i) has a lower limit value equal to or greater than 600 nm and an upper limit value equal to or less than 750 nm.

14. The system according to claim 9, wherein: The first dichroic mirror has high transmittance in a secondary first dichroic mirror transmission band, and the secondary first dichroic mirror transmission band (i) has a lower limit value equal to or greater than 700 nm and an upper limit value equal to or less than 900 nm.

15. The system according to claim 9, wherein: The first dichroic mirror has high transmittance in a plurality of different and non-overlapping transmission bands, each of which has a lower limit value equal to or greater than 350 nm and an upper limit value equal to or less than 900 nm.

16. The system of claim 9, wherein: The first dichroic mirror has high reflectivity in a first dichroic mirror reflection band, which (i) has a lower limit value equal to or greater than 300nm and an upper limit value equal to or less than 420nm, and (ii) does not overlap with the primary first dichroic mirror transmission band.

17. The system according to claim 9, wherein: The first dichroic mirror has high reflectivity in a first dichroic mirror reflection band, which (i) has a lower limit value equal to or greater than 430nm and an upper limit value equal to or less than 530nm, and (ii) does not overlap with the primary first dichroic mirror transmission band.

18. The system of claim 9, wherein: The first dichroic mirror has high reflectivity in a first dichroic mirror reflection band having a lower limit value equal to or greater than 480 nm and having an upper limit value equal to or less than 570 nm.

19. The system according to claim 9, wherein: The first dichroic mirror has high reflectivity in a first dichroic mirror reflection band having a lower limit value equal to or greater than 530 nm and having an upper limit value equal to or less than 610 nm.

20. The system of claim 9, wherein: The first dichroic mirror has high reflectivity in a first dichroic mirror reflection band having a lower limit value equal to or greater than 610 nm and having an upper limit value equal to or less than 670 nm.

21. The system of claim 9, wherein: The first dichroic mirror has high reflectivity in a first dichroic mirror reflection band having a lower limit value equal to or greater than 710 nm and having an upper limit value equal to or less than 770 nm.

22. The system of claim 9, wherein: The first dichroic mirror has high reflectivity in a plurality of different and non-overlapping first dichroic mirror reflection bands, each of the first dichroic mirror reflection bands (i) having a lower limit value equal to or greater than 350 nm and having an upper limit value equal to or less than 900 nm, and (ii) not overlapping with the primary first dichroic mirror transmission band.

23. The system of claim 1, wherein: The sample is arranged on the stage of the microscope.

24. The system of claim 1, further comprising a receiver adapted to receive the light beam reflected by the second dichroic mirror.

25. The system of claim 24, wherein: The receiver is a camera.

26. A microscope-based illumination and imaging system, comprising: The first light source; a first dichroic mirror, adapted to reflect the light beam from the first light source; a second dichroic mirror, adapted to transmit the light beam from the first light source to a sample; as well as a second light source, adapted to transmit one or more light beams having wavelengths falling within a second light source wavelength range through a tube lens of a microscope, the first dichroic mirror and the second dichroic mirror to the sample, wherein the second light source wavelength range has a lower limit value equal to or greater than 250 nm and an upper limit value equal to or less than 470 nm, and the second dichroic mirror is adapted to reflect the light beam from the sample.

27. The system of claim 26, wherein: The first dichroic mirror has high transmittance in a first dichroic mirror transmission band of at least the second light source wavelength range.

28. The system of claim 27, wherein: The transmission wavelength band of the first dichroic mirror extends above and / or below the wavelength range of the second light source.

29. The system of claim 26, wherein: The first dichroic mirror has high transmittance in a first dichroic mirror transmission band, and the first dichroic mirror transmission band has a lower limit value equal to or greater than 250 nm and an upper limit value equal to or less than 470 nm.

30. The system of claim 29, wherein: The first dichroic mirror has high reflectivity in a first dichroic mirror reflection band, and the first dichroic mirror reflection band does not overlap with the first dichroic mirror transmission band.

31. The system of claim 30, wherein: The first dichroic mirror reflection wavelength band has a lower limit value equal to or greater than 300 nm and an upper limit value equal to or less than 900 nm.

32. The system of claim 26, wherein: The second dichroic mirror has high transmittance in a second dichroic mirror transmission band of at least the second light source wavelength range.

33. The system of claim 32, wherein: The transmission wavelength band of the second dichroic mirror extends above and / or below the wavelength range of the second light source.

34. The system of claim 26, wherein: The second dichroic mirror has high transmittance in a primary second dichroic mirror transmission band having a lower limit value equal to or greater than 250 nm and having an upper limit value equal to or less than 530 nm.

35. The system of claim 34, wherein: The second dichroic mirror has high transmittance in a secondary second dichroic mirror transmission band, the secondary second dichroic mirror transmission band (i) has a lower limit value equal to or greater than 430 nm and has an upper limit value equal to or less than 530 nm and (ii) does not overlap with the primary first dichroic mirror transmission band.

36. The system of claim 34, wherein: The second dichroic mirror has high transmittance in a secondary second dichroic mirror transmission band, and the secondary second dichroic mirror transmission band has a lower limit value equal to or greater than 480 nm and an upper limit value equal to or less than 570 nm.

37. The system of claim 34, wherein: The second dichroic mirror has high transmittance in a secondary second dichroic mirror transmission band, the secondary second dichroic mirror transmission band having a lower limit value equal to or greater than 530 nm and having an upper limit value equal to or less than 610 nm.

38. The system of claim 34, wherein: The second dichroic mirror has high transmittance in a secondary second dichroic mirror transmission band, the secondary second dichroic mirror transmission band having a lower limit value equal to or greater than 610 nm and having an upper limit value equal to or less than 670 nm.

39. The system of claim 34, wherein: The second dichroic mirror has high transmittance in a secondary second dichroic mirror transmission band, the secondary second dichroic mirror transmission band having a lower limit value equal to or greater than 710 nm and having an upper limit value equal to or less than 770 nm.

40. The system of claim 34, wherein: The second dichroic mirror has high transmittance in a plurality of different and non-overlapping transmission bands, each of the transmission bands having a lower limit value equal to or greater than 400 nm and an upper limit value equal to or less than 900 nm.

41. The system of claim 34, wherein: The second dichroic mirror has high reflectivity in a second dichroic mirror reflection band that (i) has a lower limit value equal to or greater than 350 nm and an upper limit value equal to or less than 470 nm and (ii) does not overlap with the primary second dichroic mirror transmission band.

42. The system of claim 34, wherein: The second dichroic mirror has high reflectivity in a second dichroic mirror reflection band which (i) has a lower limit value equal to or greater than 440 nm and an upper limit value equal to or less than 570 nm and (ii) does not overlap with the primary second dichroic mirror transmission band.

43. The system of claim 34, wherein: The second dichroic mirror has high reflectivity in a second dichroic mirror reflection band having a lower limit value equal to or greater than 500 nm and having an upper limit value equal to or less than 650 nm.

44. The system of claim 34, wherein: The second dichroic mirror has high reflectivity in a second dichroic mirror reflection band having a lower limit value equal to or greater than 600 nm and having an upper limit value equal to or less than 750 nm.

45. The system of claim 34, wherein: The second dichroic mirror has high reflectivity in a second dichroic mirror reflection band having a lower limit value equal to or greater than 700 nm and an upper limit value equal to or less than 900 nm.

46. ​​The system of claim 34, wherein: The second dichroic mirror has high reflectivity in a plurality of different and non-overlapping second dichroic mirror reflection bands, each of which (i) has a lower limit value equal to or greater than 300 nm and an upper limit value equal to or less than 900 nm, and (ii) does not overlap with the primary second dichroic mirror transmission band.

47. The system of claim 26, wherein: The sample is arranged on the stage of the microscope.

48. The system of claim 26, further comprising a receiver adapted to receive the light beam reflected by the second dichroic mirror.

49. The system of claim 48, wherein: The receiver is a camera.

50. A method comprising: projecting a light beam from a first light source in a first light source wavelength range to a first dichroic mirror to reflect the light beam onto a sample; causing a light beam reflected by or generated by the sample in response to the first light source to travel through the first dichroic mirror to a second dichroic mirror to reflect the light beam to a receiver; and A light beam from a second light source in a second light source wavelength range is projected onto the sample through the second dichroic mirror and through the first dichroic mirror.

51. The method of claim 50, wherein: The first light source wavelength includes a first light source wavelength range.

52. The method of claim 50, wherein: The first light source wavelength comprises approximately 488 nm.

53. The method of claim 50, wherein: The first light source wavelength comprises approximately 561 nm.

54. The method of claim 50, wherein: The first light source wavelength comprises approximately 635 nm.

55. The method of claim 50, wherein: The second light source has a wavelength range of approximately 250 to 470 nm.

56. The method of claim 50, wherein: The second light source has a wavelength range suitable for excitation, photoactivation, photomanipulation or other photochemical processing of the sample.

57. The method of claim 50, wherein: The first dichroic mirror and the second dichroic mirror have high transmittance in the second light source wavelength range of the second light source.

58. The method of claim 57, wherein: The first dichroic mirror and the second dichroic mirror transmit more than 80% of the light beam from the second light source.

59. The method of claim 50, wherein: The second dichroic mirror has high reflectivity in a second dichroic mirror reflection band, and the second dichroic mirror reflection band does not overlap with the second dichroic mirror transmission band.

60. The method of claim 50, wherein: The second dichroic mirror reflection wavelength band has a lower limit value equal to or greater than 350 nm and an upper limit value equal to or less than 900 nm.

61. The method of claim 50, wherein: The first dichroic mirror has high transmittance in a first dichroic mirror transmission band of at least the second light source wavelength range.

62. The method of claim 50, wherein: The receiver is a camera.

63. A method comprising: projecting a light beam of a first light source wavelength from a first light source to a first dichroic mirror to reflect the light beam through a second dichroic mirror to a sample; causing a light beam reflected by or generated by the sample in response to the first light source to travel to the second dichroic mirror to reflect the light beam to a receiver; as well as A light beam in a second light source wavelength range from a second light source is projected onto the sample through the first dichroic mirror and through the second dichroic mirror.

64. The method of claim 63, wherein: The first light source wavelength includes a first light source wavelength range.

65. The method of claim 63, wherein: The first light source wavelength comprises approximately 488 nm.

66. The method of claim 63, wherein: The first light source wavelength comprises approximately 561 nm.

67. The method of claim 63, wherein: The first light source wavelength comprises approximately 635 nm.

68. The method of claim 63, wherein: The second light source has a wavelength range of approximately 250 to 470 nm.

69. The method of claim 63, wherein: The second light source has a wavelength range suitable for excitation, photoactivation, photomanipulation or other photochemical processing of the sample.

70. The method of claim 63, wherein: The first dichroic mirror and the second dichroic mirror have high transmittance in the second light source wavelength range of the second light source.

71. The method of claim 70, wherein: The first dichroic mirror and the second dichroic mirror transmit more than 80% of the light beam from the second light source.

72. The method of claim 63, wherein: The receiver is a camera.

73. A system according to any one of claims 1 to 49, wherein: The system does not include a mechanical switch for switching the path of light from the first light source or the second light source.

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