- Email: [email protected]
Improved Pathological Examination of Tumors with 3D Light-Sheet Microscopy
TRECAN 253 No. of Pages 4
Forum
Improved Pathological Examination of Tumors with 3D Light-Sheet Microscopy Per Uhlén1,* and Nobuyuki Tanaka1,2,* Light-sheet microscopy offers new possibilities to efficiently visualize large tissue samples in three dimensions. Volumetric 3D imaging can uncover detailed information about the inner landscape of tumors, which can improve cancer diagnosis and therapy. This Forum article highlights the advantages of using light-sheet microscopy for pathological examinations of intact tumor specimens. For decades, pathologists have used hematoxylin and eosin staining or other assistive techniques such as immunohistochemistry or in situ hybridization to examine and stage tumor sections with bright-field or fluorescence microscopy. The results of these pathological examinations are used to diagnose diseases, predict patient prognoses, and decide treatment plans. However, the assessment of tumor sections with conventional analog microscopes are laborand time-intensive processes that do not provide complete information. Histopathological examination is commonly performed on tissue samples that have been fixed and then thinly sectioned, stained, and mounted on glass slides. Usually, only a small fraction of the entire tumor is sectioned and examined under microscopy. This creates an information gap between the limited 2D images assessed and the original state of the 3D tumor, which could be vital for the assessment of diseases that are
challenging to diagnose and grade, with Fast Nondestructive Imaging of potentially significant consequences for Intact Clinical Specimens patients. Faster diagnosis and grading of pathology specimens is desirable, as compleSolid cancers exhibit complex 3D micro- tion of a pathology report can take several environments consisting of heteroge- days. In particular, rapid examinations, of neous populations of cells of various the order of 20–30 min, are essential for sizes, different genotypes, and distinct intraoperative imaging of freshly resected phenotypic characteristics [1]. This specimens for margin assessment and intratumoral heterogeneity is central to surgical guidance. The light-sheet the natural selection that drives the pro- microscopy technology offers a faster cesses of carcinogenesis, metastasis, workflow than conventional optical-secand acquired resistance to therapeutic tioning microscopy methods, such as interventions. For example, epithelial-to- confocal and nonlinear microscopy. mesenchymal transition (EMT) and angiogenesis are processes involved in creat- In a report by Glaser et al., an open-top, ing heterogeneous tumor landscapes and light-sheet microscopy system is preunique 3D masses. The ability to charac- sented that enables rapid, nondestructive terize these intratumoral features in imaging of intact clinical specimens of patient specimens would significantly arbitrary size and thickness [3] (Figure 1, help in improving the diagnosis and grad- arrow 1). The system was successfully ing of cancers and for guiding treatment used for surface microscopy of lumpecdecisions. tomy margins intraoperatively within only 1 minute after minimal processing. The More advanced light-microscopy exami- acquired digitized data provided surnations of tumor specimens are now geons with both panoramic views of tispossible due to the recent development sue surfaces and high-resolution views of of computers with high-performance relevant breast pathology. Conventional data handling capabilities and digital intraoperative pathology consultations cameras with fast sensitive digital detec- require 20–30 min to deliver a diagnosis tor arrays [2]. These technologies have and rely upon the physical sectioning of contributed to the development of new, rapidly frozen tissue specimens; a proadvanced microscopy systems, for cess that introduces tissue damage and example, the light-sheet microscope, artifacts that can lead to erroneous which enable object visualization in three diagnoses. dimensions. In the simplest form of a light-sheet microscope, the illumination Furthermore, the open-top, light-sheet light is shaped into a sheet, which allows microscopy system is used to identify rapid camera-based imaging of entire prostate carcinoma accurately from fresh frames orthogonally to the light path tissue specimens. Large fresh human (Box 1). Decoupling the illumination and prostate slices, approximately detection beam paths provides an added 3.1 3.5 0.4 cm in size were stained level of design flexibility that can enable with acridine orange and imaged within both rapid and deep volumetric imaging about 10 min per slice to identify slices of whole tissues. These advances in 3D containing carcinoma. Because laboratoimaging have also stimulated the devel- ries commonly use partial-sampling opment of novel advanced software methods to triage the number of tools for volume rendering and 3D data inspected specimens by random selecanalysis. tion or gross inspection, reducing the time
Trends in Cancer, Month Year, Vol. xx, No. yy
1
TRECAN 253 No. of Pages 4
Box 1. Light-Sheet Microscopy Light-sheet microscopy is a fluorescence microscopy technique that offers high-speed 3D sectioning [2]. The sample is illuminated with a thin sheet of light (thereby its name) of a few hundred nanometers to a few micrometers (Figure I). In contrast to regular epifluorescence microscopy the sample is illuminated perpendicularly to the direction of observation. For illumination, a laser beam is used to create either a static light sheet, or a dynamically formed light sheet by scanning a thin pencil beam in one direction, or by utilizing a dithered lattice. The light-sheet produces a 2D sheet of fluorescence that is imaged in two dimensions in the orthogonal directional by an sCMOS camera (Figure I). Because light sheets are imaged with a 2D detector array, instead of by raster scanning a single point within the tissue (as in confocal or nonlinear microscopy), light-sheet microscopes can acquire images at higher speeds. Since only a thin section is illuminated that is efficiently and fully imaged by the detector array, it reduces the photodamage and stress induced on living samples by alternative techniques such as confocal microscopy[7_TD$IF][5.
Emission light ns
e le cƟv
ExcitaƟon light je
Ob
Sa
High-speed sCMOS camera
m
ple
Figure I. Schematic Diagram Showing a Simplified Model of the Excitation Light and Emission Light in a Light-Sheet Microscope. The light sheet is projected onto the sample from the side, that is, perpendicular to the optical axis of the objective lens, hence illuminating the entire focal plane of the microscope. Abbreviation: sCMOS, scientific complementary metal-oxide-semiconductor.
needed to image tissue samples may developed and optimized for various speincrease pathological examination cific applications. For example, a protocol named CUBIC (Clear, Unobstructed Brain accuracy. Imaging Cocktail) has been applied to study cancer metastasis using wholeVolumetric Imaging of Tumor body and -organ clearing approach [5] Samples All solid tumors are unique 3D masses and another protocol named CLARITY that are shaped by the distinct interac- has been used to determine osteoprogetions between cancer cells and microen- nitors within intact bone marrow [6]. The vironments. It is not feasible to analyze open-top, light-sheet microscopy system and correlate the 3D spatial distribution of described in Glaser et al. is also used for cells and multiple proteins within intact volumetric imaging of optically cleared, tumors using conventional clinical diag- core-needle prostate biopsy specimens, nostic methods such as computed showing the ability to potentially improve tomography, magnetic resonance imag- the Gleason grading of prostate tumors ing, and histopathology. For imaging and to improve treatment decisions [3]. tumors in 3D with single-cell resolution, organic solvent-based clearing has been Volumetric 3D imaging can reveal spatial adapted [4]. Today several different tis- details regarding the constitution of whole sue-clearing protocols exist that were tumors that can be used to create new 2
Trends in Cancer, Month Year, Vol. xx, No. yy
and more efficient diagnostic readouts. Tanaka et al. presented a new pipeline termed DIPCO (Diagnosing Immunolabeled Paraffin-embedded Cleared Organs), which uses light-sheet microscopy to perform 3D examination of cleared and immunolabeled formalinfixed paraffin embedded (FFPE) tumor samples [7] (Figure 1, arrow 2). In this study, the organic solvent-based iDISCO clearing protocol [8] was used because it allowed for immunolabeling and reembedding of FFPE samples. FFPE storage of tissue specimens is standard clinical procedure for hospital biobanks worldwide. The DIPCO approach could determine tumor stage and stratify patient prognosis based on clinical samples of different cancer types with higher accuracy than current diagnostic methods.
TRECAN 253 No. of Pages 4
Specimen Fresh tumor sample
Light-sheet microscopy
Data analysis
1
Formalin-fixed paraffin-embedded Sample Cleared
3D reconstruc on
2
Light-sheet
Discovery
Clinical transla on
Cellular heterogeneity Slide-free nondestruc ve pathology Rapid intraopera ve tumor-margin examina on Environmental heterogeneity Comprehensive detailed volumetric tumor analysis
Efficient cancer staging
Figure 1. Schematic Diagram Showing the Essential Steps, Discoveries, and Clinical Translations Associated with Pathological Examinations of Tumor Samples Using Light-Sheet Microscopy. Arrow 1 indicates rapid examination of intraoperatively fresh tissue and arrow 2 indicates examination of optically cleared tissue.
Hallmarks of cancers are scrutinized in intact tumors with single-cell resolution to identify unique patterns of phenotypic heterogeneity in the features of the vasculature and EMT. 3D-data imaging analysis of the tumor vasculature provide more accurate tumor staging than conventional histological 2D methods. The DIPCO pipeline also enables
nondestructive study and diagnosis of new and old FFPE samples, as the samples can be re-embedded in paraffin after examination for future use. Tissue-clearing techniques and lightsheet microscopy have also been used to study human lung and lymph node tissues [9]. As described in Nojima
et al., performing a histopathological screening of a lymph node metastasis enabled improved sensitivity for the detection of minor metastatic carcinoma nodules in lymph node. Another study by Lee et al. utilized 3D confocal microscopy of optically cleared tissues for volumetric and multiplexed immunofluorescence imaging of tumors [10]. In this case, Trends in Cancer, Month Year, Vol. xx, No. yy
3
TRECAN 253 No. of Pages 4
cutting tumors into submillimeter macrosections enabled simple and rapid immunofluorescence labeling, optical clearing, and 3D confocal microscopy of large tissue specimens within 3 days. Here, the time frame of 3 days can be shortened if using light-sheet microscopy.
Concluding Remarks
microscopy [11] and a new clearing protocol named uDISCO (ultimate DISCO) was recently presented, demonstrating the ability to image centimeter-sized tissue samples and entire organs with lightsheet microscopy [12]. However, challenges to implementation of this new technology may exist due to the centuries-old technological framework of slidebased 2D histopathology that is currently intrenched as the gold-standard method for disease diagnosis in medicine. Nevertheless, future preclinical and clinical studies should reveal whether light-sheet microscopy and related technologies, including tissue clearing/labeling, and machine-learning approaches for computer-aided diagnosis, can fulfill its promise.
A workflow using light-sheet microscopy can potentially be implemented in clinical settings (Figure 1). Its applications in clinical pathology are diverse, including rapid surface scanning and volumetric imaging of intact specimens. These new potential uses have important implications for streamlining pathology workflows, guiding surgical oncology, and improving the pre-, intra-, and postoperative diagnosis and staging of human tumors. A better understanding of population-specific Acknowledgments cancer subtypes can help in drug devel- This study was supported by the Swedish Research opment and in designing better clinical Council (grant 2017-00815 to P.U.), the Swedish translational strategies. Cancer Society (grant CAN 2016/801 to P.U.), the David and Astrid Hagelén Foundation (N.T.), and the
New applications of light-sheet microscopy and the development of new optical clearing and fluorescence labeling protocols are frequently being reported. For example, volumetric in situ hybridization has been demonstrated with light-sheet
4
Trends in Cancer, Month Year, Vol. xx, No. yy
Karolinska Institutet Research Foundation (P.U. and N.T.). 1
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77, Stockholm, Sweden 2 Department of Urology, Keio University School of Medicine, 160-8582 Tokyo, Japan
*Correspondence: [email protected] (P. Uhlén) and [email protected] (N. Tanaka). https://doi.org/10.1016/j.trecan.2018.03.003 References 1. McGranahan, N. and Swanton, C. (2015) Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell 27, 15–26 2. Tomer, R. et al. (2013) Light sheet microscopy in cell biology. Methods Mol. Biol. 931, 123–137 3. Glaser, A.K. et al. (2017) Light-sheet microscopy for slidefree non-destructive pathology of large clinical specimens. Nat. Biomed. Eng 1, 0084 4. Dobosz, M. et al. (2014) Multispectral fluorescence ultramicroscopy: three-dimensional visualization and automatic quantification of tumor morphology, drug penetration, and antiangiogenic treatment response. Neoplasia 16, 1–13 5. Kubota, S.I. et al. (2017) Whole-body profiling of cancer metastasis with single-cell resolution. Cell Rep. 20, 236– 250 6. Greenbaum, A. et al. (2017) Bone CLARITY: clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow. Sci. Transl. Med. 9, eaah6518 7. Tanaka, N. et al. (2017) Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity. Nat. Biomed. Eng. 1, 796–806 8. Renier, N. et al. (2014) iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 9. Nojima, S. et al. (2017) CUBIC pathology: three-dimensional imaging for pathological diagnosis. Sci. Rep. 7, 9269 10. Lee, S.S. et al. (2017) Multiplex three-dimensional optical mapping of tumor immune microenvironment. Sci. Rep. 7, 17031 11. Sylwestrak, E.L. et al. (2016) Multiplexed intact-tissue transcriptional analysis at cellular resolution. Cell 164, 792–804 12. Pan, C. et al. (2016) Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat. Methods 13, 859–867
Forum
Improved Pathological Examination of Tumors with 3D Light-Sheet Microscopy Per Uhlén1,* and Nobuyuki Tanaka1,2,* Light-sheet microscopy offers new possibilities to efficiently visualize large tissue samples in three dimensions. Volumetric 3D imaging can uncover detailed information about the inner landscape of tumors, which can improve cancer diagnosis and therapy. This Forum article highlights the advantages of using light-sheet microscopy for pathological examinations of intact tumor specimens. For decades, pathologists have used hematoxylin and eosin staining or other assistive techniques such as immunohistochemistry or in situ hybridization to examine and stage tumor sections with bright-field or fluorescence microscopy. The results of these pathological examinations are used to diagnose diseases, predict patient prognoses, and decide treatment plans. However, the assessment of tumor sections with conventional analog microscopes are laborand time-intensive processes that do not provide complete information. Histopathological examination is commonly performed on tissue samples that have been fixed and then thinly sectioned, stained, and mounted on glass slides. Usually, only a small fraction of the entire tumor is sectioned and examined under microscopy. This creates an information gap between the limited 2D images assessed and the original state of the 3D tumor, which could be vital for the assessment of diseases that are
challenging to diagnose and grade, with Fast Nondestructive Imaging of potentially significant consequences for Intact Clinical Specimens patients. Faster diagnosis and grading of pathology specimens is desirable, as compleSolid cancers exhibit complex 3D micro- tion of a pathology report can take several environments consisting of heteroge- days. In particular, rapid examinations, of neous populations of cells of various the order of 20–30 min, are essential for sizes, different genotypes, and distinct intraoperative imaging of freshly resected phenotypic characteristics [1]. This specimens for margin assessment and intratumoral heterogeneity is central to surgical guidance. The light-sheet the natural selection that drives the pro- microscopy technology offers a faster cesses of carcinogenesis, metastasis, workflow than conventional optical-secand acquired resistance to therapeutic tioning microscopy methods, such as interventions. For example, epithelial-to- confocal and nonlinear microscopy. mesenchymal transition (EMT) and angiogenesis are processes involved in creat- In a report by Glaser et al., an open-top, ing heterogeneous tumor landscapes and light-sheet microscopy system is preunique 3D masses. The ability to charac- sented that enables rapid, nondestructive terize these intratumoral features in imaging of intact clinical specimens of patient specimens would significantly arbitrary size and thickness [3] (Figure 1, help in improving the diagnosis and grad- arrow 1). The system was successfully ing of cancers and for guiding treatment used for surface microscopy of lumpecdecisions. tomy margins intraoperatively within only 1 minute after minimal processing. The More advanced light-microscopy exami- acquired digitized data provided surnations of tumor specimens are now geons with both panoramic views of tispossible due to the recent development sue surfaces and high-resolution views of of computers with high-performance relevant breast pathology. Conventional data handling capabilities and digital intraoperative pathology consultations cameras with fast sensitive digital detec- require 20–30 min to deliver a diagnosis tor arrays [2]. These technologies have and rely upon the physical sectioning of contributed to the development of new, rapidly frozen tissue specimens; a proadvanced microscopy systems, for cess that introduces tissue damage and example, the light-sheet microscope, artifacts that can lead to erroneous which enable object visualization in three diagnoses. dimensions. In the simplest form of a light-sheet microscope, the illumination Furthermore, the open-top, light-sheet light is shaped into a sheet, which allows microscopy system is used to identify rapid camera-based imaging of entire prostate carcinoma accurately from fresh frames orthogonally to the light path tissue specimens. Large fresh human (Box 1). Decoupling the illumination and prostate slices, approximately detection beam paths provides an added 3.1 3.5 0.4 cm in size were stained level of design flexibility that can enable with acridine orange and imaged within both rapid and deep volumetric imaging about 10 min per slice to identify slices of whole tissues. These advances in 3D containing carcinoma. Because laboratoimaging have also stimulated the devel- ries commonly use partial-sampling opment of novel advanced software methods to triage the number of tools for volume rendering and 3D data inspected specimens by random selecanalysis. tion or gross inspection, reducing the time
Trends in Cancer, Month Year, Vol. xx, No. yy
1
TRECAN 253 No. of Pages 4
Box 1. Light-Sheet Microscopy Light-sheet microscopy is a fluorescence microscopy technique that offers high-speed 3D sectioning [2]. The sample is illuminated with a thin sheet of light (thereby its name) of a few hundred nanometers to a few micrometers (Figure I). In contrast to regular epifluorescence microscopy the sample is illuminated perpendicularly to the direction of observation. For illumination, a laser beam is used to create either a static light sheet, or a dynamically formed light sheet by scanning a thin pencil beam in one direction, or by utilizing a dithered lattice. The light-sheet produces a 2D sheet of fluorescence that is imaged in two dimensions in the orthogonal directional by an sCMOS camera (Figure I). Because light sheets are imaged with a 2D detector array, instead of by raster scanning a single point within the tissue (as in confocal or nonlinear microscopy), light-sheet microscopes can acquire images at higher speeds. Since only a thin section is illuminated that is efficiently and fully imaged by the detector array, it reduces the photodamage and stress induced on living samples by alternative techniques such as confocal microscopy[7_TD$IF][5.
Emission light ns
e le cƟv
ExcitaƟon light je
Ob
Sa
High-speed sCMOS camera
m
ple
Figure I. Schematic Diagram Showing a Simplified Model of the Excitation Light and Emission Light in a Light-Sheet Microscope. The light sheet is projected onto the sample from the side, that is, perpendicular to the optical axis of the objective lens, hence illuminating the entire focal plane of the microscope. Abbreviation: sCMOS, scientific complementary metal-oxide-semiconductor.
needed to image tissue samples may developed and optimized for various speincrease pathological examination cific applications. For example, a protocol named CUBIC (Clear, Unobstructed Brain accuracy. Imaging Cocktail) has been applied to study cancer metastasis using wholeVolumetric Imaging of Tumor body and -organ clearing approach [5] Samples All solid tumors are unique 3D masses and another protocol named CLARITY that are shaped by the distinct interac- has been used to determine osteoprogetions between cancer cells and microen- nitors within intact bone marrow [6]. The vironments. It is not feasible to analyze open-top, light-sheet microscopy system and correlate the 3D spatial distribution of described in Glaser et al. is also used for cells and multiple proteins within intact volumetric imaging of optically cleared, tumors using conventional clinical diag- core-needle prostate biopsy specimens, nostic methods such as computed showing the ability to potentially improve tomography, magnetic resonance imag- the Gleason grading of prostate tumors ing, and histopathology. For imaging and to improve treatment decisions [3]. tumors in 3D with single-cell resolution, organic solvent-based clearing has been Volumetric 3D imaging can reveal spatial adapted [4]. Today several different tis- details regarding the constitution of whole sue-clearing protocols exist that were tumors that can be used to create new 2
Trends in Cancer, Month Year, Vol. xx, No. yy
and more efficient diagnostic readouts. Tanaka et al. presented a new pipeline termed DIPCO (Diagnosing Immunolabeled Paraffin-embedded Cleared Organs), which uses light-sheet microscopy to perform 3D examination of cleared and immunolabeled formalinfixed paraffin embedded (FFPE) tumor samples [7] (Figure 1, arrow 2). In this study, the organic solvent-based iDISCO clearing protocol [8] was used because it allowed for immunolabeling and reembedding of FFPE samples. FFPE storage of tissue specimens is standard clinical procedure for hospital biobanks worldwide. The DIPCO approach could determine tumor stage and stratify patient prognosis based on clinical samples of different cancer types with higher accuracy than current diagnostic methods.
TRECAN 253 No. of Pages 4
Specimen Fresh tumor sample
Light-sheet microscopy
Data analysis
1
Formalin-fixed paraffin-embedded Sample Cleared
3D reconstruc on
2
Light-sheet
Discovery
Clinical transla on
Cellular heterogeneity Slide-free nondestruc ve pathology Rapid intraopera ve tumor-margin examina on Environmental heterogeneity Comprehensive detailed volumetric tumor analysis
Efficient cancer staging
Figure 1. Schematic Diagram Showing the Essential Steps, Discoveries, and Clinical Translations Associated with Pathological Examinations of Tumor Samples Using Light-Sheet Microscopy. Arrow 1 indicates rapid examination of intraoperatively fresh tissue and arrow 2 indicates examination of optically cleared tissue.
Hallmarks of cancers are scrutinized in intact tumors with single-cell resolution to identify unique patterns of phenotypic heterogeneity in the features of the vasculature and EMT. 3D-data imaging analysis of the tumor vasculature provide more accurate tumor staging than conventional histological 2D methods. The DIPCO pipeline also enables
nondestructive study and diagnosis of new and old FFPE samples, as the samples can be re-embedded in paraffin after examination for future use. Tissue-clearing techniques and lightsheet microscopy have also been used to study human lung and lymph node tissues [9]. As described in Nojima
et al., performing a histopathological screening of a lymph node metastasis enabled improved sensitivity for the detection of minor metastatic carcinoma nodules in lymph node. Another study by Lee et al. utilized 3D confocal microscopy of optically cleared tissues for volumetric and multiplexed immunofluorescence imaging of tumors [10]. In this case, Trends in Cancer, Month Year, Vol. xx, No. yy
3
TRECAN 253 No. of Pages 4
cutting tumors into submillimeter macrosections enabled simple and rapid immunofluorescence labeling, optical clearing, and 3D confocal microscopy of large tissue specimens within 3 days. Here, the time frame of 3 days can be shortened if using light-sheet microscopy.
Concluding Remarks
microscopy [11] and a new clearing protocol named uDISCO (ultimate DISCO) was recently presented, demonstrating the ability to image centimeter-sized tissue samples and entire organs with lightsheet microscopy [12]. However, challenges to implementation of this new technology may exist due to the centuries-old technological framework of slidebased 2D histopathology that is currently intrenched as the gold-standard method for disease diagnosis in medicine. Nevertheless, future preclinical and clinical studies should reveal whether light-sheet microscopy and related technologies, including tissue clearing/labeling, and machine-learning approaches for computer-aided diagnosis, can fulfill its promise.
A workflow using light-sheet microscopy can potentially be implemented in clinical settings (Figure 1). Its applications in clinical pathology are diverse, including rapid surface scanning and volumetric imaging of intact specimens. These new potential uses have important implications for streamlining pathology workflows, guiding surgical oncology, and improving the pre-, intra-, and postoperative diagnosis and staging of human tumors. A better understanding of population-specific Acknowledgments cancer subtypes can help in drug devel- This study was supported by the Swedish Research opment and in designing better clinical Council (grant 2017-00815 to P.U.), the Swedish translational strategies. Cancer Society (grant CAN 2016/801 to P.U.), the David and Astrid Hagelén Foundation (N.T.), and the
New applications of light-sheet microscopy and the development of new optical clearing and fluorescence labeling protocols are frequently being reported. For example, volumetric in situ hybridization has been demonstrated with light-sheet
4
Trends in Cancer, Month Year, Vol. xx, No. yy
Karolinska Institutet Research Foundation (P.U. and N.T.). 1
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-171 77, Stockholm, Sweden 2 Department of Urology, Keio University School of Medicine, 160-8582 Tokyo, Japan
*Correspondence: [email protected] (P. Uhlén) and [email protected] (N. Tanaka). https://doi.org/10.1016/j.trecan.2018.03.003 References 1. McGranahan, N. and Swanton, C. (2015) Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell 27, 15–26 2. Tomer, R. et al. (2013) Light sheet microscopy in cell biology. Methods Mol. Biol. 931, 123–137 3. Glaser, A.K. et al. (2017) Light-sheet microscopy for slidefree non-destructive pathology of large clinical specimens. Nat. Biomed. Eng 1, 0084 4. Dobosz, M. et al. (2014) Multispectral fluorescence ultramicroscopy: three-dimensional visualization and automatic quantification of tumor morphology, drug penetration, and antiangiogenic treatment response. Neoplasia 16, 1–13 5. Kubota, S.I. et al. (2017) Whole-body profiling of cancer metastasis with single-cell resolution. Cell Rep. 20, 236– 250 6. Greenbaum, A. et al. (2017) Bone CLARITY: clearing, imaging, and computational analysis of osteoprogenitors within intact bone marrow. Sci. Transl. Med. 9, eaah6518 7. Tanaka, N. et al. (2017) Whole-tissue biopsy phenotyping of three-dimensional tumours reveals patterns of cancer heterogeneity. Nat. Biomed. Eng. 1, 796–806 8. Renier, N. et al. (2014) iDISCO: a simple, rapid method to immunolabel large tissue samples for volume imaging. Cell 159, 896–910 9. Nojima, S. et al. (2017) CUBIC pathology: three-dimensional imaging for pathological diagnosis. Sci. Rep. 7, 9269 10. Lee, S.S. et al. (2017) Multiplex three-dimensional optical mapping of tumor immune microenvironment. Sci. Rep. 7, 17031 11. Sylwestrak, E.L. et al. (2016) Multiplexed intact-tissue transcriptional analysis at cellular resolution. Cell 164, 792–804 12. Pan, C. et al. (2016) Shrinkage-mediated imaging of entire organs and organisms using uDISCO. Nat. Methods 13, 859–867