BACKGROUND: Sperm metabolic pathways that generate energy for motility are compartmentalized within the flagellum. Dysfunctions in metabolic compartments, namely mitochondrial respiration and glycolysis, can compromise motility and male fertility. Studying these compartments is thus required for fertility treatment. However, it is very challenging to capture images of metabolic compartments in motile spermatozoa because the fast beating of the flagellum introduces motion blur. Therefore, most approaches immobilize spermatozoa prior to imaging. RESULTS: Our findings indicate that immobilizing sperm alters their metabolic profile, highlighting the necessity for measuring metabolism in spermatozoa during movement. We achieved this by encapsulating mouse epididymis in a hydrogel followed by two-photon fluorescence lifetime imaging microscopy for imaging motile sperm in situ. The autofluorescence of endogenous metabolites-FAD, NADH, and NADPH-enabled us to visualize sperm metabolic compartments without staining. We trained machine learning for automated image segmentation and generated metabolic fingerprints using object-based phasor analysis. We show that metabolic fingerprints of spermatozoa and the mitochondrial compartment (1) can distinguish individual males by genetic background, age, or fecundity status, (2) correlate with fertility, and (3) change with age likely due to increased oxidative metabolism. CONCLUSIONS: Our approach eliminates the need for sperm immobilization and labeling and captures the native state of sperm metabolism. This technique could be adapted for metabolism-based sperm selection for assisted reproduction.

Department of Computer Science and Artificial Intelligence University of Basque Country UPV EHU San Sebastián Spain

Imaging Methods Core Facility at BIOCEV Faculty of Science Charles University Vestec Czech Republic

Laboratory of Germ Cell Development Division BIOCEV Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Present address Department of Meiosis Max Planck Institute for Multidisciplinary Sciences Göttingen Germany

Present addresses Instituto Biofisika CSIC UPV EHU Leioa Spain

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Zecchin A, Stapor PC, Goveia J, Carmeliet P. Metabolic pathway compartmentalization: an underappreciated opportunity? Curr Opin Biotechnol. 2015;34:73–81. PubMed

Lindemann CB, Lesich KA. Functional anatomy of the mammalian sperm flagellum. Cytoskeleton (Hoboken). 2016;73(11):652–69. PubMed

Freitas MJ, Vijayaraghavan S, Fardilha M. Signaling mechanisms in mammalian sperm motility. Biol Reprod. 2017;96(1):2–12. PubMed

Ford WC. Glycolysis and sperm motility: does a spoonful of sugar help the flagellum go round? Hum Reprod Update. 2006;12(3):269–74. PubMed

du Plessis SS, Agarwal A, Mohanty G, van der Linde M. Oxidative phosphorylation versus glycolysis: what fuel do spermatozoa use? Asian J Androl. 2015;17(2):230–5. PubMed PMC

Alves LQ, Ruivo R, Valente R, Fonseca MM, Machado AM, Plon S, Monteiro N, Garcia-Parraga D, Ruiz-Diaz S, Sanchez-Calabuig MJ, et al. A drastic shift in the energetic landscape of toothed whale sperm cells. Curr Biol. 2021;31(16):3648–3655 e3649. PubMed

Fraser LR, Quinn PJ. A glycolytic product is obligatory for initiation of the sperm acrosome reaction and whiplash motility required for fertilization in the mouse. J Reprod Fertil. 1981;61(1):25–35. PubMed

Bone W, Jones NG, Kamp G, Yeung CH, Cooper TG. Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro. J Reprod Fertil. 2000;118(1):127–35. PubMed

Hoshi K, Tsukikawa S, Sato A. Importance of Ca2+, K+ and glucose in the medium for sperm penetration through the human zona pellucida. Tohoku J Exp Med. 1991;165(2):99–104. PubMed

Williams AC, Ford WC. The role of glucose in supporting motility and capacitation in human spermatozoa. J Androl. 2001;22(4):680–95. PubMed

Demain LA, Conway GS, Newman WG. Genetics of mitochondrial dysfunction and infertility. Clin Genet. 2017;91(2):199–207. PubMed

Folgero T, Bertheussen K, Lindal S, Torbergsen T, Oian P. Mitochondrial disease and reduced sperm motility. Hum Reprod. 1993;8(11):1863–8. PubMed

Carra E, Sangiorgi D, Gattuccio F, Rinaldi AM. Male infertility and mitochondrial DNA. Biochem Biophys Res Commun. 2004;322(1):333–9. PubMed

Nakada K, Sato A, Yoshida K, Morita T, Tanaka H, Inoue S, Yonekawa H, Hayashi J. Mitochondria-related male infertility. Proc Natl Acad Sci U S A. 2006;103(41):15148–53. PubMed PMC

Jiang M, Kauppila TES, Motori E, Li X, Atanassov I, Folz-Donahue K, Bonekamp NA, Albarran-Gutierrez S, Stewart JB, Larsson NG. Increased total mtDNA copy number cures male infertility despite unaltered mtDNA mutation load. Cell Metab. 2017;26(2):429–436 e424. PubMed

Rovio AT, Marchington DR, Donat S, Schuppe HC, Abel J, Fritsche E, Elliott DJ, Laippala P, Ahola AL, McNay D, et al. Mutations at the mitochondrial DNA polymerase (POLG) locus associated with male infertility. Nat Genet. 2001;29(3):261–2. PubMed

Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly YM, Gidlof S, Oldfors A, Wibom R, et al. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004;429(6990):417–23. PubMed

Takasaki N, Tachibana K, Ogasawara S, Matsuzaki H, Hagiuda J, Ishikawa H, Mochida K, Inoue K, Ogonuki N, Ogura A, et al. A heterozygous mutation of GALNTL5 affects male infertility with impairment of sperm motility. Proc Natl Acad Sci U S A. 2014;111(3):1120–5. PubMed PMC

Liu X, Li Q, Wang W, Liu F. Aberrant expression of spermspecific glycolytic enzymes are associated with poor sperm quality. Mol Med Rep. 2019;19(4):2471–8. PubMed PMC

Miki K, Qu W, Goulding EH, Willis WD, Bunch DO, Strader LF, Perreault SD, Eddy EM, O’Brien DA. Glyceraldehyde 3-phosphate dehydrogenase-S, a sperm-specific glycolytic enzyme, is required for sperm motility and male fertility. Proc Natl Acad Sci U S A. 2004;101(47):16501–6. PubMed PMC

Reynolds S, Calvert SJ, Paley MN, Pacey AA. 1H magnetic resonance spectroscopy of live human sperm. Mol Hum Reprod. 2017;23(7):441–51. PubMed PMC

Paiva C, Amaral A, Rodriguez M, Canyellas N, Correig X, Ballesca JL, Ramalho-Santos J, Oliva R. Identification of endogenous metabolites in human sperm cells using proton nuclear magnetic resonance ((1) H-NMR) spectroscopy and gas chromatography-mass spectrometry (GC-MS). Andrology. 2015;3(3):496–505. PubMed

Menezes EB, Velho ALC, Santos F, Dinh T, Kaya A, Topper E, Moura AA, Memili E. Uncovering sperm metabolome to discover biomarkers for bull fertility. BMC Genomics. 2019;20(1):714. PubMed PMC

Jensen EC. Use of fluorescent probes: their effect on cell biology and limitations. Anat Rec (Hoboken). 2012;295(12):2031–6. PubMed

Kolenc OI, Quinn KP. Evaluating cell metabolism through autofluorescence imaging of NAD(P)H and FAD. Antioxid Redox Signal. 2019;30(6):875–89. PubMed PMC

Chance B, Schoener B, Oshino R, Itshak F, Nakase Y. Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals. J Biol Chem. 1979;254(11):4764–71. PubMed

Patterson GH, Knobel SM, Arkhammar P, Thastrup O, Piston DW. Separation of the glucose-stimulated cytoplasmic and mitochondrial NAD(P)H responses in pancreatic islet beta cells. Proc Natl Acad Sci U S A. 2000;97(10):5203–7. PubMed PMC

Huang S, Heikal AA, Webb WW. Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys J. 2002;82(5):2811–25. PubMed PMC

Blinova K, Levine RL, Boja ES, Griffiths GL, Shi ZD, Ruddy B, Balaban RS. Mitochondrial NADH fluorescence is enhanced by complex I binding. Biochemistry. 2008;47(36):9636–45. PubMed PMC

Skala MC, Riching KM, Gendron-Fitzpatrick A, Eickhoff J, Eliceiri KW, White JG, Ramanujam N. In vivo multiphoton microscopy of NADH and FAD redox states, fluorescence lifetimes, and cellular morphology in precancerous epithelia. Proc Natl Acad Sci U S A. 2007;104(49):19494–9. PubMed PMC

Georgakoudi I, Quinn KP. Optical imaging using endogenous contrast to assess metabolic state. Annu Rev Biomed Eng. 2012;14:351–67. PubMed

Georgakoudi I, Quinn KP. Label-free optical metabolic imaging in cells and tissues. Annu Rev Biomed Eng. 2023;25:413–43. PubMed PMC

Lakowicz JR, Szmacinski H, Nowaczyk K, Johnson ML. Fluorescence lifetime imaging of free and protein-bound NADH. Proc Natl Acad Sci U S A. 1992;89(4):1271–5. PubMed PMC

Berezin MY, Achilefu S. Fluorescence lifetime measurements and biological imaging. Chem Rev. 2010;110(5):2641–84. PubMed PMC

Sparks H, Kondo H, Hooper S, Munro I, Kennedy G, Dunsby C, French P, Sahai E. Heterogeneity in tumor chromatin-doxorubicin binding revealed by in vivo fluorescence lifetime imaging confocal endomicroscopy. Nat Commun. 2018;9(1):2662. PubMed PMC

Becker W, Bergmann A, Hink MA, Konig K, Benndorf K, Biskup C. Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc Res Tech. 2004;63(1):58–66. PubMed

Becker W. Fluorescence lifetime imaging–techniques and applications. J Microsc. 2012;247(2):119–36. PubMed

Datta R, Heaster TM, Sharick JT, Gillette AA, Skala MC. Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications. J Biomed Opt. 2020;25(7):1–43. PubMed PMC

Serafino MJ, Jo JA. Direct frequency domain fluorescence lifetime imaging using simultaneous ultraviolet and visible excitation. Biomed Opt Express. 2023;14(4):1608–25. PubMed PMC

Konig K, So PT, Mantulin WW, Tromberg BJ, Gratton E. Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress. J Microsc. 1996;183(Pt 3):197–204. PubMed

Pattison DI, Davies MJ. Actions of ultraviolet light on cellular structures. EXS. 2006;96:131–57. PubMed

Park J, Gao L. Advancements in fluorescence lifetime imaging microscopy Instrumentation: towards high speed and 3D. Curr Opin Solid State Mater Sci. 2024;30:30. PubMed PMC

Sorrells JE, Iyer RR, Yang L, Bower AJ, Spillman DR Jr, Chaney EJ, Tu H, Boppart SA. Real-time pixelwise phasor analysis for video-rate two-photon fluorescence lifetime imaging microscopy. Biomed Opt Express. 2021;12(7):4003–19. PubMed PMC

Samimi K, Desa DE, Lin W, Weiss K, Li J, Huisken J, Miskolci V, Huttenlocher A, Chacko JV, Velten A, et al. Light-sheet autofluorescence lifetime imaging with a single-photon avalanche diode array. J Biomed Opt. 2023;28(6): 066502. PubMed PMC

Moen E, Bannon D, Kudo T, Graf W, Covert M, Van Valen D. Deep learning for cellular image analysis. Nat Methods. 2019;16(12):1233–46. PubMed PMC

Stringari C, Cinquin A, Cinquin O, Digman MA, Donovan PJ, Gratton E. Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue. Proc Natl Acad Sci U S A. 2011;108(33):13582–7. PubMed PMC

Wetzker C, Reinhardt K. Distinct metabolic profiles in Drosophila sperm and somatic tissues revealed by two-photon NAD(P)H and FAD autofluorescence lifetime imaging. Sci Rep. 2019;9(1):19534. PubMed PMC

Reinhardt K, Breunig HG, Uchugonova A, Konig K. Sperm metabolism is altered during storage by female insects: evidence from two-photon autofluorescence lifetime measurements in bedbugs. J R Soc Interface. 2015;12(110):0609. PubMed PMC

Vishnyakova P, Nikonova E, Jumaniyazova E, Solovyev I, Kirillova A, Farmakovskaya M, Savitsky A, Shirshin E, Sukhikh G, Fatkhudinov T. Fluorescence lifetime imaging microscopy as an instrument for human sperm assessment. Biochem Biophys Res Commun. 2023;645:10–6. PubMed

Cong A, Pimenta RML, Lee HB, Mereddy V, Holy J, Heikal AA. Two-photon fluorescence lifetime imaging of intrinsic NADH in three-dimensional tumor models. Cytometry A. 2019;95(1):80–92. PubMed

Norambuena A, Wallrabe H, Cao R, Wang DB, Silva A, Svindrych Z, Periasamy A, Hu S, Tanzi RE, Kim DY et al. A novel lysosome‐to‐mitochondria signaling pathway disrupted by amyloid‐ b oligomers. EMBO J. 2018;37(22):1–18. PubMed PMC

Aguilar-Arnal L, Ranjit S, Stringari C, Orozco-Solis R, Gratton E, Sassone-Corsi P. Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species. Proc Natl Acad Sci U S A. 2016;113(45):12715–20. PubMed PMC

Wright BK, Andrews LM, Markham J, Jones MR, Stringari C, Digman MA, Gratton E. NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy. Biophys J. 2012;103(1):L7–9. PubMed PMC

Winet H, Bernstein GS, Head J. Observations on the response of human spermatozoa to gravity, boundaries and fluid shear. J Reprod Fertil. 1984;70(2):511–23. PubMed

Maude AD. Non-random distribution of bull spermatozoa in a drop of sperm suspension. Nature. 1963;200:381. PubMed

Woolley DM. Motility of spermatozoa at surfaces. Reproduction. 2003;126(2):259–70. PubMed

Tourmente M, Varea-Sanchez M, Roldan ERS. Faster and more efficient swimming: energy consumption of murine spermatozoa under sperm competitiondagger. Biol Reprod. 2019;100(2):420–8. PubMed

Digman MA, Caiolfa VR, Zamai M, Gratton E. The phasor approach to fluorescence lifetime imaging analysis. Biophys J. 2008;94(2):L14–16. PubMed PMC

Nakashima N, Yoshihara K, Tanaka F, Yagi K. Picosecond fluorescence lifetime of the coenzyme of D-amino acid oxidase. J Biol Chem. 1980;255(11):5261–3. PubMed

Buffone MG, Ijiri TW, Cao W, Merdiushev T, Aghajanian HK, Gerton GL. Heads or tails? Structural events and molecular mechanisms that promote mammalian sperm acrosomal exocytosis and motility. Mol Reprod Dev. 2012;79(1):4–18. PubMed PMC

Kuang W, Zhang J, Lan Z, Deepak R, Liu C, Ma Z, Cheng L, Zhao X, Meng X, Wang W, et al. SLC22A14 is a mitochondrial riboflavin transporter required for sperm oxidative phosphorylation and male fertility. Cell Rep. 2021;35(3): 109025. PubMed PMC

Ferramosca A, Zara V. Bioenergetics of mammalian sperm capacitation. Biomed Res Int. 2014;2014: 902953. PubMed PMC

Orgebin-Crist MC. Sperm maturation in rabbit epididymis. Nature. 1967;216(5117):816–8. PubMed

Rosenthal N, Brown S. The mouse ascending: perspectives for human-disease models. Nat Cell Biol. 2007;9(9):993–9. PubMed

Cinco R, Digman MA, Gratton E, Luderer U. Spatial characterization of bioenergetics and metabolism of primordial to preovulatory follicles in whole ex vivo murine ovary. Biol Reprod. 2016;95(6):129. PubMed PMC

Renaud O, Herbomel P, Kissa K. Studying cell behavior in whole zebrafish embryos by confocal live imaging: application to hematopoietic stem cells. Nat Protoc. 2011;6(12):1897–904. PubMed

Cao R, Wallrabe H, Periasamy A. Multiphoton FLIM imaging of NAD(P)H and FAD with one excitation wavelength. J Biomed Opt. 2020;25(1):1–16. PubMed PMC

Ma N, Mochel NR, Pham PD, Yoo TY, Cho KWY, Digman MA. Label-free assessment of pre-implantation embryo quality by the Fluorescence Lifetime Imaging Microscopy (FLIM)-phasor approach. Sci Rep. 2019;9(1):13206. PubMed PMC

Walsh AJ, Mueller KP, Tweed K, Jones I, Walsh CM, Piscopo NJ, Niemi NM, Pagliarini DJ, Saha K, Skala MC. Classification of T-cell activation via autofluorescence lifetime imaging. Nat Biomed Eng. 2021;5(1):77–88. PubMed PMC

Stringari C, Wang H, Geyfman M, Crosignani V, Kumar V, Takahashi JS, Andersen B, Gratton E. In vivo single-cell detection of metabolic oscillations in stem cells. Cell Rep. 2015;10(1):1–7. PubMed PMC

Olaf Ronneberger PF, Thomas Brox: U-Net: convolutional networks for biomedical image segmentation. 2015. Preprint at: https://arxiv.org/abs/1505.04597.

Tourmente M, Rowe M, Gonzalez-Barroso MM, Rial E, Gomendio M, Roldan ER. Postcopulatory sexual selection increases ATP content in rodent spermatozoa. Evolution. 2013;67(6):1838–46. PubMed

Tourmente M, Villar-Moya P, Rial E, Roldan ER. Differences in ATP generation via glycolysis and oxidative phosphorylation and relationships with sperm motility in mouse species. J Biol Chem. 2015;290(33):20613–26. PubMed PMC

Tourmente M, Villar-Moya P, Varea-Sanchez M, Luque-Larena JJ, Rial E, Roldan ER. Performance of rodent spermatozoa over time is enhanced by increased ATP concentrations: the role of sperm competition. Biol Reprod. 2015;93(3):64. PubMed

Albrechtova J, Albrecht T, Dureje L, Pallazola VA, Pialek J. Sperm morphology in two house mouse subspecies: do wild-derived strains and wild mice tell the same story? PLoS ONE. 2014;9(12): e115669. PubMed PMC

Gage MJ, Macfarlane CP, Yeates S, Ward RG, Searle JB, Parker GA. Spermatozoal traits and sperm competition in Atlantic salmon: relative sperm velocity is the primary determinant of fertilization success. Curr Biol. 2004;14(1):44–7. PubMed

Birkhead TR, Martinez JG, Burke T, Froman DP. Sperm mobility determines the outcome of sperm competition in the domestic fowl. Proc Biol Sci. 1999;266(1430):1759–64. PubMed PMC

Kahrl AF, Snook RR, Fitzpatrick JL. Fertilization mode differentially impacts the evolution of vertebrate sperm components. Nat Commun. 2022;13(1):6809. PubMed PMC

Samplaski MK, Nangia AK. Adverse effects of common medications on male fertility. Nat Rev Urol. 2015;12(7):401–13. PubMed

Sakkas D, Ramalingam M, Garrido N, Barratt CL. Sperm selection in natural conception: what can we learn from Mother Nature to improve assisted reproduction outcomes? Hum Reprod Update. 2015;21(6):711–26. PubMed PMC

Leung ETY, Lee CL, Tian X, Lam KKW, Li RHW, Ng EHY, Yeung WSB, Chiu PCN. Simulating nature in sperm selection for assisted reproduction. Nat Rev Urol. 2022;19(1):16–36. PubMed

Kusari F, Mihola O, Schimenti J, Trachtulec Z. Meiotic Epigenetic Factor PRDM9 Impacts Sperm Quality of Hybrid Mice. Reprod. 2020;160(1):53–64. PubMed

Mihola O, Kobets T, Krivankova K, Linhartova E, Gasic S, Schimenti JC, Trachtulec Z. Copy-number variation introduced by long transgenes compromises mouse male fertility independently of pachytene checkpoints. Chromosoma. 2020;129(1):69–82. PubMed

Sun N, Youle RJ, Finkel T. The mitochondrial basis of aging. Mol Cell. 2016;61(5):654–66. PubMed PMC

Lopez-Otin C, Blasco MA, Partridge L, Serrano M, Kroemer G. The hallmarks of aging. Cell. 2013;153(6):1194–217. PubMed PMC

Aitken RJ. Impact of oxidative stress on male and female germ cells: implications for fertility. Reproduction. 2020;159(4):R189–201. PubMed

He L, He T, Farrar S, Ji L, Liu T, Ma X. Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species. Cell Physiol Biochem. 2017;44(2):532–53. PubMed

Twigg J, Fulton N, Gomez E, Irvine DS, Aitken RJ. Analysis of the impact of intracellular reactive oxygen species generation on the structural and functional integrity of human spermatozoa: lipid peroxidation, DNA fragmentation and effectiveness of antioxidants. Hum Reprod. 1998;13(6):1429–36. PubMed

Almeida S, Rato L, Sousa M, Alves MG, Oliveira PF. Fertility and sperm quality in the aging male. Curr Pharm Des. 2017;23(30):4429–37. PubMed

Selvaratnam J, Robaire B. Overexpression of catalase in mice reduces age-related oxidative stress and maintains sperm production. Exp Gerontol. 2016;84:12–20. PubMed

Ozkosem B, Feinstein SI, Fisher AB, O’Flaherty C. Advancing age increases sperm chromatin damage and impairs fertility in peroxiredoxin 6 null mice. Redox Biol. 2015;5:15–23. PubMed PMC

Paoli D, Pecora G, Pallotti F, Faja F, Pelloni M, Lenzi A, Lombardo F. Cytological and molecular aspects of the ageing sperm. Hum Reprod. 2019;34(2):218–27. PubMed

Cocuzza M, Athayde KS, Agarwal A, Sharma R, Pagani R, Lucon AM, Srougi M, Hallak J. Age-related increase of reactive oxygen species in neat semen in healthy fertile men. Urology. 2008;71(3):490–4. PubMed

Iaffaldano N, Di Iorio M, Mannina L, Paventi G, Rosato MP, Cerolini S, Sobolev AP. Age-dependent changes in metabolic profile of turkey spermatozoa as assessed by NMR analysis. PLoS ONE. 2018;13(3): e0194219. PubMed PMC

Losano J, Angrimani D, Dalmazzo A, Rui BR, Brito MM, Mendes CM, Kawai G, Vannucchi CI, Assumpcao M, Barnabe VH, et al. Effect of mitochondrial uncoupling and glycolysis inhibition on ram sperm functionality. Reprod Domest Anim. 2017;52(2):289–97. PubMed

Saggiorato G, Alvarez L, Jikeli JF, Kaupp UB, Gompper G, Elgeti J. Human sperm steer with second harmonics of the flagellar beat. Nat Commun. 2017;8(1):1415. PubMed PMC

Becker W, Braun L, Suarez-Ibarrol R, Miernik A. Metabolic IMAGING BY SIMULTANEous FLIM of NAD(P)H and FAD. Current Directions in Biomedical Engineering. 2020;6(3):254–6.

Gregorova S, Forejt J. PWD/Ph and PWK/Ph inbred mouse strains of Mus m. musculus subspecies–a valuable resource of phenotypic variations and genomic polymorphisms. Folia Biol (Praha). 2000;46(1):31–41. PubMed

Sun Y, Day RN, Periasamy A. Investigating protein-protein interactions in living cells using fluorescence lifetime imaging microscopy. Nat Protoc. 2011;6(9):1324–40. PubMed PMC

Durmaz AR, Muller M, Lei B, Thomas A, Britz D, Holm EA, Eberl C, Mucklich F, Gumbsch P. A deep learning approach for complex microstructure inference. Nat Commun. 2021;12(1):6272. PubMed PMC

Pan C, Schoppe O, Parra-Damas A, Cai R, Todorov MI, Gondi G, von Neubeck B, Bogurcu-Seidel N, Seidel S, Sleiman K, et al. Deep learning reveals cancer metastasis and therapeutic antibody targeting in the entire body. Cell. 2019;179(7):1661–1676 e1619. PubMed PMC

Nygate YN, Levi M, Mirsky SK, Turko NA, Rubin M, Barnea I, Dardikman-Yoffe G, Haifler M, Shalev A, Shaked NT. Holographic virtual staining of individual biological cells. Proc Natl Acad Sci U S A. 2020;117(17):9223–31. PubMed PMC

Manifold B, Men S, Hu R, Fu D. A versatile deep learning architecture for classification and label-free prediction of hyperspectral images. Nat Mach Intell. 2021;3:306–15. PubMed PMC

Glastonbury CA, Pulit SL, Honecker J, Censin JC, Laber S, Yaghootkar H, Rahmioglu N, Pastel E, Kos K, Pitt A, et al. Machine learning based histology phenotyping to investigate the epidemiologic and genetic basis of adipocyte morphology and cardiometabolic traits. PLoS Comput Biol. 2020;16(8): e1008044. PubMed PMC

Kingma DP, Ba J: Adam: a method for stochastic optimization. 2014. Preprint at: https://arxiv.org/abs/1412.6980.

He K, Zhang X, Ren S, Sun J: Delving deep into rectifiers: surpassing human-level performance on ImageNet classification. IEEE International Conference on Computer Vision (ICCV). 2015:1026-1034.

Abadi M. TensorFlow: learning functions at scale. ACM SIGPLAN Notices. 2016;51(9):1–1.

Kuhn HW: The Hungarian method for the assignment problem. Naval research logistics quarterly. 1955.

Soltanian-Zadeh S, Sahingur K, Blau S, Gong Y, Farsiu S. Fast and robust active neuron segmentation in two-photon calcium imaging using spatiotemporal deep learning. Proc Natl Acad Sci U S A. 2019;116(17):8554–63. PubMed PMC

Giovannucci A, Friedrich J, Gunn P, Kalfon J, Brown BL, Koay SA, Taxidis J, Najafi F, Gauthier JL, Zhou P, et al. CaImAn an open source tool for scalable calcium imaging data analysis. Elife. 2019;8:8. PubMed PMC

Ranjit S, Malacrida L, Jameson DM, Gratton E. Fit-free analysis of fluorescence lifetime imaging data using the phasor approach. Nat Protoc. 2018;13(9):1979–2004. PubMed

R Development Core Team. R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing; 2019.

Midway S, Robertson M, Flinn S, Kaller M. Comparing multiple comparisons: practical guidance for choosing the best multiple comparisons test. PeerJ. 2020;8: e10387. PubMed PMC