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US-12618846-B2 - Method and device for imaging fluorescent proteins in near- and short-wave infrared

US12618846B2US 12618846 B2US12618846 B2US 12618846B2US-12618846-B2

Abstract

The present invention relates to systems, methods and fluorescent polypeptide for real-time multicolor shortwave infrared fluorescence imaging. The systems and methods of the present invention further relate to real-time multi-color in vivo SWIR imaging systems employing high-power excitation sources in combination with state of the art InGaAs SWIR detectors and SWIR illuminated fluorescent polypeptide.

Inventors

  • Oliver Bruns
  • Jakob LINGG
  • Juan-Pablo FUENZALIDA-WERNER
  • Andre STIEL
  • Martin WARMER
  • Shyam S. RAMAKRISHNAN
  • Emily COSCO
  • Ellen Sletten

Assignees

  • Helmholtz Zentrum München—Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH)
  • THE REGENTS OF THE UNIVERSITY OF CALIFORNIA

Dates

Publication Date
20260505
Application Date
20200607

Claims (16)

  1. 1 . A system for imaging, comprising: i) a biological sample comprising a fluorescent polypeptide expressed therein, wherein the fluorescent polypeptide comprises an excitation wavelength that causes the fluorescent polypeptide to emit a fluorescence signal comprising a tail portion in the near infrared (NIR) wavelength range of the electromagnetic spectrum and/or in the shortwave infrared (SWIR) wavelength range of electromagnetic spectrum; ii) an excitation source configured to emit electromagnetic radiation comprising the excitation wavelength; and iii) a detector configured to detect the tail portion of the fluorescence signal emitted by of the fluorescent polypeptide, wherein said detector is configured to detect in the near infrared (NIR) wavelength range of the electromagnetic spectrum and/or in the shortwave infrared (SWIR) wavelength range of electromagnetic spectrum.
  2. 2 . The system of claim 1 , further comprising a computing device, wherein the detector outputs a detected tail portion signal from the fluorescent probe to the computing device.
  3. 3 . The system of claim 2 , wherein the computing device compares the detected fluorescence signal to an intensity threshold to a subject condition.
  4. 4 . The system of claim 1 , further comprising a display, wherein the detector is configured to outputs the detected tail portion of the fluorescence signal emitted by the fluorescent polypeptide to the display.
  5. 5 . The system of claim 1 , wherein said fluorescent polypeptide is excitable at a wavelength of less than about 800 nm.
  6. 6 . The system of claim 1 , wherein said fluorescent polypeptide comprises one or more of the following polypeptides: i) a fluorescent polypeptide which is at least 60% or more identical to tdTomato polypeptide (SEQ ID NO: 1); ii) a fluorescent polypeptide which is at least 60% or more identical to SMURF polypeptide (SEQ ID NO: 2); iii) a fluorescent polypeptide which is at least 60% or more identical to iRFP720 polypeptide (SEQ ID NO: 3); iv) a fluorescent polypeptide which is at least 60% or more identical to 22G (Dronpa) from Echinophyllia sp. SC22, Genbank ADE48854.1, v) a fluorescent polypeptide which is at least 60% or more identical to aceGFP from Aequorea coerulescens , Genbank AAN41637, vi) a fluorescent polypeptide which is at least 60% or more identical to amFP486 from Anemonia majano , Genbank AAF03371, vii) a fluorescent polypeptide which is at least 60% or more identical to anm2CP from Anthoathecata, Genbank AAR85352, viii) a fluorescent polypeptide which is at least 60% or more identical to avGFP (classic GFP) from Aequorea victoria , Genbank AAA27721, ix) a fluorescent polypeptide which is at least 60% or more identical to cFP484 from Clavularia sp., Genbank AAF03374, x) a fluorescent polypeptide which is at least 60% or more identical to dendFP from Dendronephthya sp., Genbank AAM10625, xi) a fluorescent polypeptide which is at least 60% or more identical to dfGFP from Olindias formosus , Genbank BBC28143, xii) a fluorescent polypeptide which is at least 60% or more identical to DrCBD from Deinococcus radiodurans , Genbank AE001825, xiii) a fluorescent polypeptide which is at least 60% or more identical to DsRed from Discosoma sp., Genbank AAF03369, xiv) a fluorescent polypeptide which is at least 60% or more identical to EosFP from Lobophyllia hemprichii , Genbank AAV54099, xv) a fluorescent polypeptide which is at least 60% or more identical to eqFP578 from Entacmaea quadricolor , Genbank H3JQU7, xvi) a fluorescent polypeptide which is at least 60% or more identical to eqFP611 from Entacmaea quadricolor , Genbank AAN05449, xvii) a fluorescent polypeptide which is at least 60% or more identical to HcRed from Heteractis crispa , Genbank Q95W85.1, xviii) a fluorescent polypeptide which is at least 60% or more identical to KikG from Favia favus , Genbank BAD95670.1, xix) a fluorescent polypeptide which is at least 60% or more identical to KO from Verrillofungia concinna , Genbank BAD24721, xx) a fluorescent polypeptide which is at least 60% or more identical to LanYFP from Branchiostoma lanceolatum , Genbank ACA48232, xxi) a fluorescent polypeptide which is at least 60% or more identical to SEQ ID NO: 4, xxii) a fluorescent polypeptide which is at least 60% or more identical to mRed7 having SEQ ID NO: 5, xxiii) a fluorescent polypeptide which is at least 60% or more identical to pR3784g from Nostoc punctiforme , Genbank WP_012410140, xxiv) a fluorescent polypeptide which is at least 60% or more identical to RpBphP1 from Rhodopseudomonas palustris , Genbank 5OY5_A, XXV) a fluorescent polypeptide which is at least 60% or more identical to RpBphP2 from Rhodopseudomonas palustris , Genbank WP_011158562, xxvi) a fluorescent polypeptide which is at least 60% or more identical to RpBphP6 from Rhodopseudomonas palustris , Genbank WP_011156523, xxvii) a fluorescent polypeptide which is at least 60% or more identical to TeAPCalpha from Trichodesmium erythraeum IMS101, Genbank CP000393.1, xxviii) a fluorescent polypeptide which is at least 60% or more identical to zFP538 from Zoanthus sp., Genbank AAF03373, xxix) a fluorescent polypeptide which is at least 60% or more identical to BphP AGP1 from Agrobacterium tumefaciens , Genbank F7UC55_RHIRD, xxx) a fluorescent polypeptide which is at least 60% or more identical to sGPC2 from Acaryochloris marina (Chee et al., Journal of Biomedical Optics 23 (10), 106006 (October 2018)), xxxi) a fluorescent polypeptide which is at least 60% or more identical to APCF2 from Chroococcidiopsis thermalis , Genbank WP_015153831, xxxii) a fluorescent polypeptide which is at least 60% or more identical to UnaG from Anguilla japonica , Genbank AB763906, xxxiii) a fluorescent polypeptide which is at least 60% or more identical to RRvT polypeptide (SEQ ID NO: 6), xxxiv) a fluorescent polypeptide which is at least 60% or more identical to tdTomato polypeptide (SEQ ID NO: 7), XXXV) a fluorescent polypeptide which is at least 60% or more identical to tdimer2(12) polypeptide (SEQ ID NO: 8), xxxvi) a fluorescent polypeptide which is at least 60% or more identical to pcDronpa2 polypeptide (SEQ ID NO: 9), xxxvii) a fluorescent polypeptide which is at least 60% or more identical to mScarlet polypeptide (SEQ ID NO: 10), xxxviii) a fluorescent polypeptide which is at least 60% or more identical to mKO kappa polypeptide (SEQ ID NO: 11), xxxix) a fluorescent polypeptide which is at least 60% or more identical to TurboRFP polypeptide (SEQ ID NO: 12), xl) a fluorescent polypeptide which is at least 60% or more identical to PsmOrange polypeptide (SEQ ID NO: 13), xli) a fluorescent polypeptide which is at least 60% or more identical to RFP611 polypeptide (SEQ ID NO: 14), xlii) a fluorescent polypeptide which is at least 60% or more identical to mRuby3 polypeptide (SEQ ID NO: 15), xliii) a fluorescent polypeptide which is at least 60% or more identical to vsfGFP-0 polypeptide (SEQ ID NO: 16), xliv) a fluorescent polypeptide which is at least 60% or more identical to bfloGFPa1 polypeptide (Bomati et al. (2014). Scientific Reports, 4 (1), 5469, doi: 10.1038/srep05469), xlv) a fluorescent polypeptide which is at least 60% or more identical to LanYFP polypeptide (SEQ ID NO: 17), xlvi) a fluorescent polypeptide which is at least 60% or more identical to dLanYFP polypeptide (SEQ ID NO: 18), xlvii) a fluorescent polypeptide which is at least 60% or more identical to dVFP polypeptide (SEQ ID NO: 19), xlviii) a fluorescent polypeptide which is at least 60% or more identical to ccalYFP polypeptide (SEQ ID NO: 20), xlix) a fluorescent polypeptide which is at least 60% or more identical to efasGFP polypeptide (SEQ ID NO: 21), l) A fluorescent polypeptide which is at least 60% or more identical to pcDronpa (green) polypeptide (SEQ ID NO: 22), li) a fluorescent polypeptide which is at least 60% or more identical to aeurGFP polypeptide (SEQ ID NO: 23), lii) a fluorescent polypeptide which is at least 60% or more identical to miRFP720 polypeptide (SEQ ID NO: 24), liii) a fluorescent polypeptide which is at least 60% or more identical to iRFP720 polypeptide (SEQ ID NO: 25), liv) a fluorescent polypeptide which is at least 60% or more identical to Wi-Phy polypeptide (SEQ ID NO: 26), lv) a fluorescent polypeptide which is at least 60% or more identical to SNIFP polypeptide (SEQ ID NO: 27), lvi) a fluorescent polypeptide which is at least 60% or more identical to iFP2.0 polypeptide (SEQ ID NO: 28), lvii) a fluorescent polypeptide which is at least 60% or more identical to iRFP713 polypeptide (SEQ ID NO: 29), lviii) a fluorescent polypeptide which is at least 60% or more identical to iFP1.4 polypeptide (SEQ ID NO: 30), lix) a fluorescent polypeptide which is at least 60% or more identical to mIFP polypeptide (SEQ ID NO: 31), lx) a fluorescent polypeptide which is at least 60% or more identical to miRFP709 polypeptide (SEQ ID NO: 32), lxi) a fluorescent polypeptide which is at least 60% or more identical to miRFP polypeptide (SEQ ID NO: 33), lxii) a fluorescent polypeptide which is at least 60% or more identical to M35NA polypeptide (SEQ ID NO: 34), lxiii) a fluorescent polypeptide which is at least 60% or more identical to smURFP polypeptide (SEQ ID NO: 35), lxiv) a fluorescent polypeptide which is at least 60% or more identical to TDsmURFP polypeptide (SEQ ID NO: 36), lxv) a fluorescent polypeptide which is at least 60% or more identical to LanFP2 polypeptide (SEQ ID NO: 37), lxvi) a fluorescent polypeptide which is at least 60% or more identical to HcRed-Tandem polypeptide (SEQ ID NO: 38), lxvii) a fluorescent polypeptide which is at least 60% or more identical to Skylan-S polypeptide (SEQ ID NO: 39), lxviii) a fluorescent polypeptide which is at least 60% or more identical to VFP polypeptide (SEQ ID NO: 40), lxix) a fluorescent polypeptide which is at least 60% or more identical to GFPxm163 polypeptide (SEQ ID NO: 41), lxx) a fluorescent polypeptide which is at least 60% or more identical to PlamGFP polypeptide (SEQ ID NO: 42), lxxi) a fluorescent polypeptide which is at least 60% or more identical to sarcGFP polypeptide (SEQ ID NO: 43), lxxii) a fluorescent polypeptide which is at least 60% or more identical to psamCFP polypeptide (SEQ ID NO: 44), lxxiii) a fluorescent polypeptide which is at least 60% or more identical to GFPxm18 polypeptide (SEQ ID NO: 45), lxxiv) a fluorescent polypeptide which is at least 60% or more identical to Gamillus 0.2 polypeptide (SEQ ID NO: 46), lxxv) a fluorescent polypeptide which is at least 60% or more identical to eGFP polypeptide (SEQ ID NO: 47), lxxvi) a fluorescent polypeptide which is at least 60% or more identical to eYFP polypeptide (SEQ ID NO: 48), lxxvii) a fluorescent polypeptide which is at least 60% or more identical to Venus polypeptide (SEQ ID NO: 49), lxxviii) a fluorescent polypeptide which is at least 60% or more identical to mOrange2 polypeptide (SEQ ID NO: 50), lxxix) a fluorescent polypeptide which is at least 60% or more identical to mCherry polypeptide (SEQ ID NO: 51), lxxx) a fluorescent polypeptide which is at least 60% or more identical to mTagBFP polypeptide (SEQ ID NO: 52), lxxxi) a fluorescent polypeptide which is at least 60% or more identical to ZsGreen polypeptide (SEQ ID NO: 53), lxxxii) a fluorescent polypeptide which is at least 60% or more identical to YPet polypeptide (SEQ ID NO: 54), lxxxiii) a fluorescent polypeptide which is at least 60% or more identical to mCitrine polypeptide (SEQ ID NO: 55), lxxxiv) a fluorescent polypeptide which is at least 60% or more identical to CFP polypeptide (SEQ ID NO: 56), lxxxv) a fluorescent polypeptide which is at least 60% or more identical to eCFP polypeptide (SEQ ID NO: 57), lxxxvi) a fluorescent polypeptide which is at least 60% or more identical to GFP polypeptide (SEQ ID NO: 58), lxxxvii) a fluorescent fragment of any one of (i)-(Ixxxvi).
  7. 7 . The system of claim 1 , wherein said sample expresses two or more different fluorescent polypeptides.
  8. 8 . The system of claim 1 , wherein said system is a system for multiplexed and/or multicolor imaging of said biological sample.
  9. 9 . The system of claim 1 , wherein: the excitation source comprises a first laser light source configured to operate at a first wavelength; and at least a second laser light source configured to operate at a second wavelength; and the system further comprises a control unit operably coupled to the first laser light source, the second laser light source and the detector, wherein the control unit is configured to control the first laser light source to provide a first excitation light pulse/s and to control the second laser light source to provide a second excitation light pulse/s in sequential manner; wherein the control unit is further operably coupled to the detector to switch detector in a sequential manner between a first state during which the imaging device is responsive to a first electromagnetic radiation and a second state during which the imaging device is responsive to a second electromagnetic radiation, wherein said first and second electromagnetic radiations are not identical; and wherein the system is configured such that the switching of the imaging device into the first state is triggered by the provision of the light pulse/s.
  10. 10 . A system according to claim 1 , wherein the system is configured to perform one or more of the following: i) an in vivo, ex vivo and/or in vitro method; ii) a diagnostic, therapeutic, surgical and/or screening method; iii) a tissue engineering and/or transplantation method; iv) a three-dimensional (3D) bioprinting method; v) a real-time imaging method; vi) a fluorescence imaging method; vii) a multicolor real-time image acquisition method; viii) a method for reduction of melanin absorption in the SWIR; ix) a method for a non-invasive imaging of tissues and/or organisms in the presence of melanin.
  11. 11 . A method for imaging a biological sample comprising: i) exposing at least a portion of said biological sample comprising a fluorescent polypeptide expressed therein, wherein the fluorescent polypeptide comprises an excitation wavelength that causes the fluorescent polypeptide to emit a fluorescence signal comprising a tail portion in the near infrared (NIR) wavelength range of the electromagnetic spectrum and/or in the shortwave infrared (SWIR) wavelength range of electromagnetic spectrum; and ii) detecting the tail portion of the fluorescence signal emitted by the fluorescent polypeptide, wherein said detecting is carried out in the near infrared (NIR) wavelength range of the electromagnetic spectrum and/or in the shortwave infrared (SWIR) wavelength range of the electromagnetic spectrum.
  12. 12 . The method of claim 11 , wherein said fluorescent polypeptide is excitable at a wavelength of less than about 800 nm.
  13. 13 . The method of claim 11 , wherein said fluorescent polypeptide comprises one or more of the following polypeptides: i) a fluorescent polypeptide which is at least 60% or more identical to tdTomato polypeptide (SEQ ID NO: 1); ii) a fluorescent polypeptide which is at least 60% or more identical to SMURF polypeptide (SEQ ID NO: 2); iii) a fluorescent polypeptide which is at least 60% or more identical to iRFP720 polypeptide (SEQ ID NO: 3); iv) a fluorescent polypeptide which is at least 60% or more identical to 22G (Dronpa) from Echinophyllia sp. SC22, Genbank ADE48854.1, v) a fluorescent polypeptide which is at least 60% or more identical to aceGFP from Aequorea coerulescens , Genbank AAN41637, vi) a fluorescent polypeptide which is at least 60% or more identical to amFP486 from Anemonia majano , Genbank AAF03371, vii) a fluorescent polypeptide which is at least 60% or more identical to anm2CP from Anthoathecata, Genbank AAR85352, viii) a fluorescent polypeptide which is at least 60% or more identical to avGFP (classic GFP) from Aequorea victoria , Genbank AAA27721, ix) a fluorescent polypeptide which is at least 60% or more identical to cFP484 from Clavularia sp., Genbank AAF03374, x) a fluorescent polypeptide which is at least 60% or more identical to dendFP from Dendronephthya sp., Genbank AAM10625, xi) a fluorescent polypeptide which is at least 60% or more identical to dfGFP from Olindias formosus , Genbank BBC28143, xii) a fluorescent polypeptide which is at least 60% or more identical to DrCBD from Deinococcus radiodurans , Genbank AE001825, xiii) a fluorescent polypeptide which is at least 60% or more identical to DsRed from Discosoma sp., Genbank AAF03369, xiv) a fluorescent polypeptide which is at least 60% or more identical to EosFP from Lobophyllia hemprichii , Genbank AAV54099, xv) a fluorescent polypeptide which is at least 60% or more identical to eqFP578 from Entacmaea quadricolor , Genbank H3JQU7, xvi) a fluorescent polypeptide which is at least 60% or more identical to eqFP611 from Entacmaea quadricolor , Genbank AAN05449, xvii) a fluorescent polypeptide which is at least 60% or more identical to HcRed from Heteractis crispa , Genbank Q95W85.1, xviii) a fluorescent polypeptide which is at least 60% or more identical to KikG from Favia favus , Genbank BAD95670.1, xix) a fluorescent polypeptide which is at least 60% or more identical to KO from Verrillofungia concinna , Genbank BAD24721, xx) a fluorescent polypeptide which is at least 60% or more identical to Lan YFP from Branchiostoma lanceolatum , Genbank ACA48232, xxi) a fluorescent polypeptide which is at least 60% or more identical to SEQ ID NO: 4, xxii) a fluorescent polypeptide which is at least 60% or more identical to mRed7 having SEQ ID NO: 5, xxiii) a fluorescent polypeptide which is at least 60% or more identical to pR3784g from Nostoc punctiforme , Genbank WP_012410140, xxiv) a fluorescent polypeptide which is at least 60% or more identical to RpBphP1 from Rhodopseudomonas palustris , Genbank 5OY5_A, xxv) a fluorescent polypeptide which is at least 60% or more identical to RpBphP2 from Rhodopseudomonas palustris , Genbank WP_011158562, xxvi) a fluorescent polypeptide which is at least 60% or more identical to RpBphP6 from Rhodopseudomonas palustris , Genbank WP_011156523, xxvii) a fluorescent polypeptide which is at least 60% or more identical to TeAPCalpha from Trichodesmium erythraeum IMS101, Genbank CP000393.1, xxviii) a fluorescent polypeptide which is at least 60% or more identical to zFP538 from Zoanthus sp., Genbank AAF03373, xxix) a fluorescent polypeptide which is at least 60% or more identical to BphP AGP1 from Agrobacterium tumefaciens , Genbank F7UC55_RHIRD, xxx) a fluorescent polypeptide which is at least 60% or more identical to sGPC2 from Acaryochloris marina (Chee et al., Journal of Biomedical Optics 23 (10), 106006 (October 2018)), xxxi) a fluorescent polypeptide which is at least 60% or more identical to APCF2 from Chroococcidiopsis thermalis , Genbank WP_015153831, xxxii) a fluorescent polypeptide which is at least 60% or more identical to UnaG from Anguilla japonica , Genbank AB763906, xxxiii) a fluorescent polypeptide which is at least 60% or more identical to RRvT polypeptide (SEQ ID NO: 6), xxxiv) a fluorescent polypeptide which is at least 60% or more identical to tdTomato polypeptide (SEQ ID NO: 7), xxxv) a fluorescent polypeptide which is at least 60% or more identical to tdimer2 (12) polypeptide (SEQ ID NO: 8), xxxvi) a fluorescent polypeptide which is at least 60% or more identical to pcDronpa2 polypeptide (SEQ ID NO: 9), xxxvii) a fluorescent polypeptide which is at least 60% or more identical to mScarlet polypeptide (SEQ ID NO: 10), xxxviii) a fluorescent polypeptide which is at least 60% or more identical to mKO kappa polypeptide (SEQ ID NO: 11), xxxix) a fluorescent polypeptide which is at least 60% or more identical to TurboRFP polypeptide (SEQ ID NO: 12), xl) a fluorescent polypeptide which is at least 60% or more identical to PsmOrange polypeptide (SEQ ID NO: 13), xli) a fluorescent polypeptide which is at least 60% or more identical to RFP611 polypeptide (SEQ ID NO: 14), xlii) a fluorescent polypeptide which is at least 60% or more identical to mRuby3 polypeptide (SEQ ID NO: 15), xliii) a fluorescent polypeptide which is at least 60% or more identical to vsfGFP-0 polypeptide (SEQ ID NO: 16), xliv) a fluorescent polypeptide which is at least 60% or more identical to bfloGFPa1 polypeptide (Bomati et al. (2014). Scientific Reports, 4 (1), 5469. doi: 10.1038/srep05469), xlv) a fluorescent polypeptide which is at least 60% or more identical to Lan YFP polypeptide (SEQ ID NO: 17), xlvi) a fluorescent polypeptide which is at least 60% or more identical to dLanYFP polypeptide (SEQ ID NO: 18), xlvii) a fluorescent polypeptide which is at least 60% or more identical to dVFP polypeptide (SEQ ID NO: 19), xlviii) a fluorescent polypeptide which is at least 60% or more identical to ccalYFP polypeptide (SEQ ID NO: 20), xlix) a fluorescent polypeptide which is at least 60% or more identical to efasGFP polypeptide (SEQ ID NO: 21), l) A fluorescent polypeptide which is at least 60% or more identical to pcDronpa (green) polypeptide (SEQ ID NO: 22), li) a fluorescent polypeptide which is at least 60% or more identical to aeurGFP polypeptide (SEQ ID NO: 23), lii) a fluorescent polypeptide which is at least 60% or more identical to miRFP720 polypeptide (SEQ ID NO: 24), liii) a fluorescent polypeptide which is at least 60% or more identical to iRFP720 polypeptide (SEQ ID NO: 25), liv) a fluorescent polypeptide which is at least 60% or more identical to Wi-Phy polypeptide (SEQ ID NO: 26), lv) a fluorescent polypeptide which is at least 60% or more identical to SNIFP polypeptide (SEQ ID NO: 27), lvi) a fluorescent polypeptide which is at least 60% or more identical to iFP2.0 polypeptide (SEQ ID NO: 28), lvii) a fluorescent polypeptide which is at least 60% or more identical to iRFP713 polypeptide (SEQ ID NO: 29), lviii) a fluorescent polypeptide which is at least 60% or more identical to iFP1.4 polypeptide (SEQ ID NO: 30), lix) a fluorescent polypeptide which is at least 60% or more identical to mIFP polypeptide (SEQ ID NO: 31), lx) a fluorescent polypeptide which is at least 60% or more identical to miRFP709 polypeptide (SEQ ID NO: 32), lxi) a fluorescent polypeptide which is at least 60% or more identical to miRFP polypeptide (SEQ ID NO: 33), lxii) a fluorescent polypeptide which is at least 60% or more identical to M35NA polypeptide (SEQ ID NO: 34), lxiii) a fluorescent polypeptide which is at least 60% or more identical to smURFP polypeptide (SEQ ID NO: 35), lxiv) a fluorescent polypeptide which is at least 60% or more identical to TDsmURFP polypeptide (SEQ ID NO: 36), lxv) a fluorescent polypeptide which is at least 60% or more identical to LanFP2 polypeptide (SEQ ID NO: 37), lxvi) a fluorescent polypeptide which is at least 60% or more identical to HcRed-Tandem polypeptide (SEQ ID NO: 38), lxvii) a fluorescent polypeptide which is at least 60% or more identical to Skylan-S polypeptide (SEQ ID NO: 39), lxviii) a fluorescent polypeptide which is at least 60% or more identical to VFP polypeptide (SEQ ID NO: 40), lxix) a fluorescent polypeptide which is at least 60% or more identical to GFPxm163 polypeptide (SEQ ID NO: 41), lxx) a fluorescent polypeptide which is at least 60% or more identical to PlamGFP polypeptide (SEQ ID NO: 42), lxxi) a fluorescent polypeptide which is at least 60% or more identical to sarcGFP polypeptide (SEQ ID NO: 43), lxxii) a fluorescent polypeptide which is at least 60% or more identical to psamCFP polypeptide (SEQ ID NO: 44), lxxiii) a fluorescent polypeptide which is at least 60% or more identical to GFPxm18 polypeptide (SEQ ID NO: 45), lxxiv) a fluorescent polypeptide which is at least 60% or more identical to Gamillus 0.2 polypeptide (SEQ ID NO: 46), lxxv) a fluorescent polypeptide which is at least 60% or more identical to eGFP polypeptide (SEQ ID NO: 47), lxxvi) a fluorescent polypeptide which is at least 60% or more identical to eYFP polypeptide (SEQ ID NO: 48), lxxvii) a fluorescent polypeptide which is at least 60% or more identical to Venus polypeptide (SEQ ID NO: 49), lxxviii) a fluorescent polypeptide which is at least 60% or more identical to mOrange2 polypeptide (SEQ ID NO: 50), lxxix) a fluorescent polypeptide which is at least 60% or more identical to mCherry polypeptide (SEQ ID NO: 51), lxxx) a fluorescent polypeptide which is at least 60% or more identical to mTagBFP polypeptide (SEQ ID NO: 52), lxxxi) a fluorescent polypeptide which is at least 60% or more identical to ZsGreen polypeptide (SEQ ID NO: 53), lxxxii) a fluorescent polypeptide which is at least 60% or more identical to YPet polypeptide (SEQ ID NO: 54), lxxxiii) a fluorescent polypeptide which is at least 60% or more identical to mCitrine polypeptide (SEQ ID NO: 55), lxxxiv) a fluorescent polypeptide which is at least 60% or more identical to CFP polypeptide (SEQ ID NO: 56), lxxxv) a fluorescent polypeptide which is at least 60% or more identical to eCFP polypeptide (SEQ ID NO: 57), lxxxvi) a fluorescent polypeptide which is at least 60% or more identical to GFP polypeptide (SEQ ID NO: 58), lxxxvii) a fluorescent fragment of any one of (i)-(Ixxxvi).
  14. 14 . The method of claim 11 , wherein said fluorescent probe comprises two or more different fluorescent polypeptides, preferably said two or more different fluorescent polypeptides are according.
  15. 15 . The method of claim 11 , wherein said method is the method for multiplexed and/or multicolor imaging of said biological sample.
  16. 16 . The method of claim 11 , wherein: the at least a portion of said biological sample comprising a fluorescent polypeptide expressed therein further comprises a second fluorescent polypeptide expressed therein, said suitable excitation source is configured to provide a first light pulse/s having the excitation wavelength causing the fluorescent polypeptide in the portion of said biological sample to emit the fluorescence signal, and at least a second light pulse/s having a second wavelength which is different from the first wavelength configured to cause the second fluorescent polypeptide in the portion of said biological sample to emit a second fluorescence signal comprising a second tail portion in the near infrared (NIR) wavelength range of the electromagnetic spectrum and/or in the shortwave infrared (SWIR) wavelength range of electromagnetic spectrum; wherein the first light pulse/s and the second (and/or subsequent) light pulse/s are provided sequentially; iii) detecting the tail portion of the fluorescent signal and the second fluorescence signal by an imaging device, wherein the peak emission wavelength of at least one polypeptide in the portion of said biological sample lies outside of the detection range of the imaging device, the detection process including: aa) switching the imaging device, in a sequential manner, between a first configuration during which the imaging device is responsive to a first electromagnetic radiation and a second configuration during which the imaging device is responsive to a second electromagnetic radiation, wherein said first and second electromagnetic radiations are not identical; wherein the switching of the first configuration is triggered by the provision of the light pulse.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present invention is filed under 35 U.S.C. § 371 as the U.S. national phase of International Application No. PCT/EP2020/065754, filed Jun. 7, 2020, which designated the United States and which claims the right of priority of U.S. Provisional Application No. 62/858,461, filed Jun. 7, 2019. The entire disclosures of the above-identified priority applications are hereby fully incorporated herein by reference. The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 7, 2021, is named SCH-5200-US SeqListing.txt and is 144 kilobytes in size. TECHNICAL FIELD The shortwave infrared (SWIR, e.g., in the range 1000-2000 nm) region of the electromagnetic spectrum has provided a means to real-time monitoring of whole mammals with high contrast and resolution. While many inorganic and organic fluorophores have been developed for this region, multiplexed experiments have been limited due to near infrared (NIR, e.g., in the range 700-1000 nm) excitation wavelengths of often broad and overlapping absorption profiles. The present invention relates to systems, methods and fluorescent polypeptide for real-time multicolor shortwave infrared fluorescence imaging. The systems and methods of the present invention further relate to real-time multi-color in vivo SWIR imaging systems employing high-power excitation sources in combination with state of the art InGaAs SWIR detectors and SWIR illuminated fluorescent polypeptide. The present invention further relates to fluorescence imaging that is an essential technology in biological research. The principle concept of fluorescence imaging requires a (labelled) fluorescent biological sample, an optical setup (microscopic, mesoscopic or macroscopic) for detection and an excitation light source (FIG. 17). BACKGROUND OF THE INVENTION There exist systems that are capable of performing in vivo SWIR imaging (e.g., WO2017160639A1). The indium gallium arsenide (InGaAs) detectors are restricted for commercial use and are bound by law enforcement services due to their applications in military surveillance and weapon defense systems. These detectors also lag behind in commercial development due to the high associated development cost. However, there exist a number of commercially available high-throughput InGaAs detector-based camera systems. The diode-based VIS (visible light), NIR or SWIR light sources are a mature technology, however the high power current driven VIS, NIR or SWIR light sources are safety critical apparatus and there exist relatively smaller number of system developers and service providers. The recent developments in this industry has resulted fiber-coupled light-sources with dedicated current controllable driver units. The trigger control devices are common apparatus used for imaging in visible spectrum. However, there is no known system that provides complete integration of high-power VIS/NIR/SWIR light sources with SWIR detectors for the purpose of in vivo imaging of biological structures. Prevailing in vivo real-time multicolor optical imaging systems employ visible or near-infrared spectrum for fluorescence imaging. When applied to characterize biological structures, such imaging apparatus provide sub-standard results due to higher photon scattering in biological tissues as opposed to the short-wave-infrared (SWIR) imaging systems. The short-wave-infrared imaging techniques provide better contrast and clarity in imaging due to higher transmission through biological tissues and reduced autofluorescence. However, the existing SWIR imaging systems are not capable of synthesizing a multicolor real-time in vivo imaging (e.g., acquiring 25 frames per second and faster) of biological structures. The excitation sources and detectors are not capable of handling external control for synchronized acquisition. The HDR imaging of biological structures is limited in existing SWIR imaging device and methods due to low throughput design of detectors. The controllability and scalability of the existing SWIR imaging apparatus are limited. Additionally, a real-time multi-channel fluorescence imaging system (e.g., acquiring 25 frames per second and faster) in SWIR spectrum is not yet available for commercial use due to the technical challenges faced in the development of high-throughput SWIR detectors and SWIR targeted fluorophores. On the other hand, every year imaging devices are sold for microscopic imaging (e.g., Zeiss, Leica, Olympus etc.) and for macroscopic whole-mouse imaging (e.g., Perkin Elmer, etc.). The price of those devices could, for example, range from 100000 € (e.g., epifluorescence microscope) to 1000000 € (e.g., multiphoton and surgical microscopes) and higher. The global microscopy market has been estimated to reach up to 5756 billion $ in 2019 (https://www.statista.com/statistics/523127/world-